Metabolism Of Imidacloprid Research Articles

Differential metabolism of imidacloprid and dinotefuran by Bemisia tabaci CYP6CM1 variants

Imidacloprid has been used to control one of most serious pests, Bemisia tabaci. However, B. tabaci has developed imidacloprid resistance mainly by over-expressing CYP6CM1. It was reported that imidacloprid-resistant B. tabaci showed no or low level of cross-resistance against dinotefuran. Here, we expressed CYP6CM1 variants using Sf9/baculovirus and/or Drosophila S2 cells and showed that CYP6CM1 variants metabolized imidacloprid but not dinotefuran. In addition, we demonstrated that imidacloprid and pymetrozine competed for a CYP6CM1 variant more efficiently than dinotefuran, using a luminescent substrate competition assay. These results suggest that lack of metabolic activity of CYP6CM1 variants against dinotefuran caused no or low level of cross-resistance.

The metabolism distribution and effect of imidacloprid in chinese lizards (Eremias argus) following oral exposure

Systematically evaluation of the metabolism, distribution and effect of imidacloprid in Chinese lizards (Eremias argus) were carried out following oral exposure. Imidacloprid-olefin-guanidine was prone to accumulate in the brain and caused potential neurotoxicity. Percutaneous and excretory excretions were the primary ways for the elimination of imidacloprid and its metabolites. Liver was the main site for hydroxy reduction and nitro-reduction metabolism of imidacloprid. The metabolism of imidacloprid was a complex process in which many metabolic enzymes participated. Aldehyde oxidase and CYP2C9 were the key enzymes in nitro-reduction process. CYP3A4 dominated the process of hydroxylation and desaturation. The increase in Glutathione S-transferase expression may be related to the removal of imidacloprid, but also related to the oxidative stress reaction that imidacloprid may cause in tissues, especially in the kidney. The findings enrich and supplement the knowledge of the environmental fate of imidacloprid in reptiles.

The metabolism of imidacloprid by aldehyde oxidase contributes to its clastogenic effect in New Zealand rabbits

Imidacloprid (IMI) is a systemic, chloro-nicotinyl insecticide classified in Regulation N° 1272/2008 of the European Commision as “harmful if swallowed and very toxic to aquatic life, with long-lasting effects”. IMI is metabolized in vitro both by aldehyde oxidase (AOX) (reduction) and by cytochrome P450s enzymes (CYPs). In the present study, the AOX inhibitor sodium tungstate dihydrate (ST) was used to elucidate the relative contribution of CYP 450 and AOX metabolic pathways on IMI metabolism, in male rabbits exposed to IMI for two months. To evaluate the inhibition effectiveness, various metabolite concentrations in the IMI and IMI + ST exposed groups were monitored. DNA damage was also evaluated in micronucleus (MN) and single cell electrophoresis (SCGC) assays in both groups, along with oxidative stress (OS) with the inflammatory status of the exposed animals, in order to clarify which metabolic pathway is more detrimental in this experimental setting. A significant increase in the frequency of binucleated cells with MN (BNMN, 105%) and micronuclei (MN, 142%) was observed after exposure to IMI (p < 0.001). The increase in the ST co-exposed animals was less pronounced (BNMN 75%, MN 95%). The Cytokinesis Block Proliferation Index (CBPI) showed no significant difference between controls and exposed animals at any time of exposure (p > 0.05), which indicates no cytotoxic effect. Similarly, comet results show that the IMI group exhibited the highest achieved tail intensity, which reached 70.7% over the control groups, whereas in the IMI + ST groups the increase remained at 48.5%. No differences were observed between all groups for oxidative-stress biomarkers. The results indicate that the AOX metabolic pathway plays a more important role in the systemic toxicity of IMI.

Transcriptome analysis and identification of P450 genes relevant to imidacloprid detoxification in Bradysia odoriphaga

Pesticide tolerance poses many challenges for pest control, particularly for destructive pests such as Bradysia odoriphaga. Imidacloprid has been used to control B. odoriphaga since 2013, however, imidacloprid resistance in B. odoriphaga has developed in recent years. Identifying actual and potential genes involved in detoxification metabolism of imidacloprid could offer solutions for controlling this insect. In this study, RNA-seq was used to explore differentially expressed genes in B. odoriphaga that respond to imidacloprid treatment. Differential expression data between imidacloprid treatment and the control revealed 281 transcripts (176 with annotations) showing upregulation and 394 transcripts (235 with annotations) showing downregulation. Among them, differential expression levels of seven P450 unigenes were associated with imidacloprid detoxification mechanism, with 4 unigenes that were upregulated and 3 unigenes that were downregulated. The qRT-PCR results of the seven differential expression P450 unigenes after imidacloprid treatment were consistent with RNA-Seq data. Furthermore, oral delivery mediated RNA interference of these four upregulated P450 unigenes followed by an insecticide bioassay significantly increased the mortality of imidacloprid-treated B. odoriphaga. This result indicated that the four upregulated P450s are involved in detoxification of imidacloprid. This study provides a genetic basis for further exploring P450 genes for imidacloprid detoxification in B. odoriphaga.

Partitioning the roles of CYP6G1 and gut microbes in the metabolism of the insecticide imidacloprid in Drosophila melanogaster

Resistance to insecticides through enhanced metabolism is a worldwide problem. The Cyp6g1 gene of the vinegar fly, Drosophila melanogaster, is a paradigm for the study of metabolic resistance. Constitutive overexpression of this gene confers resistance to several classes of insecticides, including the neonicotinoid imidacloprid (IMI). The metabolism of IMI in this species has been previously shown to yield oxidative and nitro-reduced metabolites. While levels of the oxidative metabolites are correlated with CYP6G1 expression, nitro-reduced metabolites are not, raising the question of how these metabolites are produced. Some IMI metabolites are known to be toxic, making their fate within the insect a second question of interest. These questions have been addressed by coupling the genetic tools of gene overexpression and CRISPR gene knock-out with the mass spectrometric technique, the Twin-Ion Method (TIM). Analysing axenic larvae indicated that microbes living within D. melanogaster are largely responsible for the production of the nitro-reduced metabolites. Knock-out of Cyp6g1 revealed functional redundancy, with some metabolites produced by CYP6G1 still detected. IMI metabolism was shown to produce toxic products that are not further metabolized but readily excreted, even when produced in the Central Nervous System (CNS), highlighting the significance of transport and excretion in metabolic resistance.

RNA interference of three up-regulated transcripts associated with insecticide resistance in an imidacloprid resistant population of Leptinotarsa decemlineata

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is a major agricultural pest of potatoes in the Central Sands production region of Wisconsin. Previous studies have shown that populations of L. decemlineata have become resistant to many classes of insecticides, including the neonicotinoid insecticide, imidacloprid. Furthermore, L. decemlineata has multiple mechanisms of resistance to deal with a pesticide insult, including enhanced metabolic detoxification by cytochrome p450s and glutathione S-transferases. With recent advances in the transcriptomic analysis of imidacloprid susceptible and resistant L. decemlineata populations, it is possible to investigate the role of candidate genes involved in imidacloprid resistance. A recently annotated transcriptome analysis of L. decemlineata was obtained from select populations of L. decemlineata collected in the Central Sands potato production region, which revealed a subset of mRNA transcripts constitutively up-regulated in resistant populations. We hypothesize that a portion of the up-regulated transcripts encoding for genes within the resistant populations also encode for pesticide resistance and can be suppressed to re-establish a susceptible phenotype. In this study, a discrete set of three up-regulated targets were selected for RNA interference experiments using a resistant L. decemlineata population. Following the successful suppression of transcripts encoding for a cytochrome p450, a cuticular protein, and a glutathione synthetase protein in a select L. decemlineata population, we observed reductions in measured resistance to imidacloprid that strongly suggest these genes control essential steps in imidacloprid metabolism in these field populations.

Persistence and Metabolism of Imidacloprid After Seed Treatment in Cotton Field Soil

A field study was carried out to determine the persistence and metabolism of imidacloprid in soil following its seed treatment @ 3.5 g and 14 g a.i. kg−1 of seed. Soil samples were collected at different time intervals from 7–90 days after application and the residues of imidacloprid and its metabolites (6-chloronicotinic acid, imidacloprid-nitrogunidine, olefin, nitrosimine, urea and 5-hydroxy) were quantified by high-performance liquid chromatography. The total imidacloprid residues in soil were mainly constituted by the parent compound followed by olefin and nitrosimine. The limit of quantification for the analysis of imidacloprid and its metabolites was worked out to be 0.01 mg kg−1. The maximum residues of total imidacloprid in soil samples collected 7 days after seed treatment were found to be 1.59 and 3.39 mg kg−1for the two dosages, rspectively. These residues could not be detected after 60 days for both the dosages. Nitrosamine metabolite was found to be the maximum followed by olefin. The half-life values (t 1/2) of total imidacloprid were calculated to be 23.89 and 26.65 days, respectively, when applied at recommended dose and four times the recommended dose, respectively.

The roles of CYP6AY1 and CYP6ER1 in imidacloprid resistance in the brown planthopper: Expression levels and detoxification efficiency

Two P450 monooxygenase genes, CYP6AY1 and CYP6ER1, were reported to contribute importantly to imidacloprid resistance in the brown planthopper, Nilaparvata lugens. Although recombinant CYP6AY1 could metabolize imidacloprid efficiently, the expression levels of CYP6ER1 gene were higher in most resistant populations. In the present study, three field populations were collected from different countries, and the bioassay, RNAi and imidacloprid metabolism were performed to evaluate the importance of two P450s in imidacloprid resistance. All three populations, DOT (Dongtai) from China, CNA (Chainat) from Thailand and HCM (Ho Chi Minh) from Vietnam, showed high resistance to imidacloprid (57.0-, 102.9- and 89.0-fold). CYP6AY1 and CYP6ER1 were both over expressed in three populations, with highest ratio of 13.2-fold for CYP6ER1 in HCM population. Synergism test and RNAi analysis confirmed the roles of both P450 genes in imidacloprid resistance. However, CYP6AY1 was indicated more important in CNA population, and CYP6AY1 and CYP6ER1 were equal in HCM population, although the expression level of CYP6ER1 (13.2-fold) was much higher than that of CYP6AY1 (4.11-fold) in HCM population. Although the recombinant proteins of both P450 genes could metabolize imidacloprid efficiently, the catalytic activity of CYP6AY1 (Kcat=3.627pmol/min/pmol P450) was significantly higher than that of CYP6ER1 (Kcat=2.785pmol/min/pmol P450). It was supposed that both P450 proteins were important for imidacloprid resistance, in which CYP6AY1 metabolized imidacloprid more efficiently and CYP6ER1 gene could be regulated by imidacloprid to a higher level.

Sex-related effects of imidacloprid modulated by piperonyl butoxide and menadione in rats. Part II: genotoxic and cytotoxic potential

Despite its intended use, imidacloprid causes genotoxic and cytotoxic effects in mammals, especially in the presence of metabolic activation systems. The aim of this study was to determine to which extent these effects are sex related and how its metabolism modulators piperonyl butoxide and menadione affect its toxicity. Male and female Sprague-Dawley rats were injected with the intraperitoneal LD50 dose of imidacloprid alone (170 mg/kg) or pretreated with piperonyl butoxide (100 mg/kg) and menadione (25 mg/kg) for 12 and 24 h. Structural chromosome aberrations, abnormal cells and mitotic index were determined microscopically in bone marrow cells. Male rats showed susceptibility to the genotoxic effects of imidacloprid. Piperonyl butoxide was effective in countering this effect only at 24 h, whereas menadione exacerbated imidacloprid-induced genotoxicity. Piperonyl butoxide and menadione pretreatments increased the percentage of structural chromosome aberrations and abnormal cells in females. Imidacloprid decreased the mitotic index, whereas pretreatment with piperonyl butoxide and menadione showed improvement in both sexes. We believe that CYP450-mediated metabolism of imidacloprid is under the hormonal control and therefore that its genotoxicity is sex related. Piperonyl butoxide pretreatment also showed sex-related modulation. The hormonal effects on imidacloprid biotransformation require further investigation.

Assessment of imidacloprid degradation by soil-isolated Bacillus alkalinitrilicus.

Imidacloprid is extensively used on a broad range of crops worldwide as seed dressing, soil treatment, and foliar application. Hence, the degradation potential of bacterial strains from sugarcane-growing soils was studied in liquid medium for subsequent use in bioremediation of contaminated soils. The microbe cultures degrading imidacloprid were isolated and enriched on Dorn's broth containing imidacloprid as sole carbon source maintained at 28°C and Bacillus alkalinitrilicus showed maximum potential to degrade imidacloprid. Clay loam soil samples were fortified with imidacloprid at 50, 100, and 150mgkg(-1) along with 45 × 10(7) microbe cells under two opposing sets of conditions, viz., autoclaved and unautoclaved. To study degradation and metabolism of imidacloprid under these two conditions, samples were drawn at regular intervals of 7, 14, 28, 35, 42, 49, and 56days. Among metabolites, three metabolites were detected, viz., 6-chloronicotinic acid, nitrosimine followed by imidacloprid-NTG under both the conditions. Total imidacloprid residues were not found to follow the first-order kinetics in both types of conditions. This paper reports for the first time the potential use of pure cultures of soil-isolated native bacterium B. alkalinitrilicus and also its use along with natural soil microflora for remediation of imidacloprid-contaminated soils.

Dissecting the Insect Metabolic Machinery Using Twin Ion Mass Spectrometry: A Single P450 Enzyme Metabolizing the Insecticide Imidacloprid in Vivo

Insecticide resistance is one of the most prevalent examples of anthropogenic genetic change, yet our understanding of metabolic-based resistance remains limited by the analytical challenges associated with rapidly tracking the in vivo metabolites of insecticides at nonlethal doses. Here, using twin ion mass spectrometry analysis of the extracts of whole Drosophila larvae and excreta, we show that (i) eight metabolites of the neonicotinoid insecticide, imidacloprid, can be detected when formed by susceptible larval genotypes and (ii) the specific overtranscription of a single gene product, Cyp6g1, associated with the metabolic resistance to neonicotinoids, results in a significant increase in the formation of three imidacloprid metabolites that are formed in C-H bond activation reactions; that is, Cyp6g1 is directly linked to the enhanced metabolism of imidacloprid in vivo. These results establish a rapid and sensitive method for dissecting the metabolic machinery of insects by directly linking single gene products to insecticide metabolism.

Impacts of Trunk and Soil Injections of Low Rates of Imidacloprid on Hemlock Woolly Adelgid (Hemiptera: Adelgidae) and Eastern Hemlock (Pinales: Pinaceae) Health

Eastern hemlocks (Tsuga canadensis Carrière) at two sites in southwestern Virginia were treated by trunk and soil injections of imidacloprid to determine the insecticide's impact on hemlock woolly adelgid, Adelges tsugae Annand. Treatments were 25, 50, and 100% of the highest labeled dosage rates for both stem and soil injection. Three and 4 yr after treatment, the half and full rates had significantly reduced A. tsugae populations, which were accompanied by increased new hemlock shoot growth and higher hemlock health scores on a visual rating of tree appearance. Imidacloprid and metabolite concentrations in tissue of treated trees were determined by liquid chromatography-tandem mass spectrometry, and A. tsugae density decreased as imidacloprid concentrations increased in wood tissue. There were no observed A. tsugae populations in all trees with imidacloprid tissue concentrations >413 ppb. Olefin, di-hydroxy, and 6-chloro-nicotinic-acid metabolites were the imidacloprid metabolites recovered in the highest concentrations. This suggests that hemlock metabolism of imidacloprid may increase efficacy of the parent compound. Stem and soil treatments of low rates of systemic imidacloprid reduce adelgid populations and promote hemlock health, but still may provide a remnant food source for beneficial predators.

Persistence and Metabolism of Imidacloprid in Rice

Imidacloprid is a systemic insecticide which gives effective control of plant and leaf hoppers in rice. The persistence and metabolism of imidacloprid in paddy leaves, rice grains, bran, straw and husk were studied following two applications of imidacloprid (Confidor 17.8 SL) @ 20 and 80g a.i. ha(-1) at an interval of 10days. Samples of paddy leaves were collected at various time intervals. The samples of rice grains, bran, straw and husk were collected at the time of harvest. The limit of quantification of imidacloprid and its metabolites was worked out to be 0.01mgkg(-1). The maximum residues of imidacloprid and its metabolites in paddy leaves after 0day (1h after last spray) of its application @ 20 and 80g a.i. ha(-1) were found to be 4.57 and 13.94mgkg(-1), respectively. These residues could not be detected after 60 and 90days following last application of imidacloprid at lower and higher dosages, respectively. In rice, olefin metabolite was found to be the main constituent, followed by nitroguanidine, urea, 5-hydroxy, chloronicotinic acid and nitrosimine. The samples of rice grains, bran, straw and husk did not reveal the presence of imidacloprid or its metabolites following its application at both the dosages at harvest.

Spatial and Temporal Distribution of Residues of Imidacloprid and Its Insecticidal 5-Hydroxy and Olefin and Metabolites in Eastern Hemlock (Pinales: Pinaceae) in the Southern Appalachians

Widespread mortality of eastern hemlock, Tsuga canadensis (L.) Carrière, resulting from infestation by hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), has occurred throughout the native range of eastern hemlock within the eastern United States. Imidacloprid, a systemic insecticide, is one of the primary chemical compounds used to control hemlock woolly adelgid in both urban and, in a limited manner, in natural forest environments. The metabolism of imidacloprid in eastern hemlock produces 12 metabolites; two of these, imidacloprid 5-hydroxy and imidacloprid olefin, are considered toxicologically important metabolites. However, little is known about the persistence of these metabolites in eastern hemlock in the southern Appalachians. Concentrations ofimidacloprid, olefin, and 5-hydroxy were quantified by using HPLC/MS/MS techniques. Over the 3-yr study, concentrations of imidacloprid and consequent 5-hydroxy and olefin were highest in trees treated with a soil injection in the spring. Imidacloprid and 5-hydroxy concentrations in sap were highest at 12 mo posttreatment and in tissue at 15 mo posttreatment. Imidacloprid was detected through 36 mo posttreatment and 5-hydroxy was detected through 15 mo posttreatment. Olefin concentrations in both sap and tissue were highest at 36 mo posttreatment and were detected in high concentrations through 36 mo posttreatment. Concentrations of imidacloprid were highest in the bottom stratum of the canopy and lowest in the top stratum. Concentrations of olefin and 5-hydroxy were highest in the top stratum and lowest in the bottom stratum.

Persistence behaviour of imidacloprid and its metabolites in soil under sugarcane

The persistence and metabolism of imidacloprid in soil under sugarcane were studied following application of imidacloprid at 20 and 80 g active ingredient (a.i.) ha(-1). Soil samples were collected at different time intervals (7, 15, 30, 45, 60 and 90 days after application), and the residues of imidacloprid and its metabolites (6-chloronicotinic acid, nitrosimine, imidacloprid-NTG, olefin, urea and 5-hydroxy) were quantified by high-performance liquid chromatography. In soil, the total imidacloprid residues were mainly constituted by the parent compound followed by 6-chloronicotinic acid, nitrosimine and imidacloprid-NTG metabolites. Maximum residues of imidacloprid and its metabolites were 4.29 and 7.81 mg kg(-1) in soil samples collected 7 days after the application of imidacloprid at 20 and 80 g a.i. ha(-1), respectively. At both doses, these residues declined to below the detectable limit in soil after 90 days of application. Olefin, urea and 5-hydroxy metabolites were not detected in soil. Dissipation of total imidacloprid residues did not follow the first-order kinetics with a coefficient of determination value of 0.883 and 0.838 for the recommended dose and four times the recommended dose, respectively. The half-life (T(1/2)) value of total imidacloprid was observed to be 10.64 and 10.10 days for the recommended dose and four times the recommended dose, respectively.

Persistence of imidacloprid and its major metabolites in sugarcane leaves and juice following its soil application

The persistence and metabolism of imidacloprid in sugarcane leaves and juice were studied following application of imidacloprid @ 20 and 80 g a.i. ha−1. Samples of sugarcane leaves were collected at various time intervals (7, 15, 30, 45, 60 and 90 days after treatment), whereas those of juice were collected at the time of harvest. The residues of imidacloprid and its metabolites (6-chloronicotinic acid, nitrosimine, imidacloprid-NTG, olefin, urea and 5-hydroxy) were quantified by high-performance liquid chromatograph. In leaves, the total imidacloprid residues were mainly constituted by the parent compound followed by 6-chloronicotinic acid metabolite. Total residues of imidacloprid and its metabolites were found to be 4.97 and 12.99 mg kg−1 in sugarcane leaves collected 7 days after the application of imidacloprid @ 20 and 80 g a.i. ha−1 respectively. At both the doses, these residues declined to below the detectable limit in the leaves after 90 days of application with the half-life values of 9.68 and 8.14 days of imidacloprid @ 20 and 80 g a.i. ha−1 respectively. The total toxic residues of imidacloprid in sugarcane leaves were 0.90 and 6.29 mg kg−1 at 7 days after treatment of imidacloprid @ 20 and 80 g a.i. ha−1 respectively. The bioconcentration factor in sugarcane leaves was 0.35 and 1.03 in the lower and higher dose respectively at 7 days after treatment. The total residues followed the pseudo first order kinetics with R2 of 0.9243 and 0.944 for recommended dose and four times the recommended dose, respectively. The residues of imidacloprid and its metabolites were not detected in samples of sugarcane juice.

Concentration Levels of Imidacloprid and Dinotefuran in Five Tissue Types of Black Walnut, Juglans nigra

Black walnut, a valuable economic and environmentally important species, is threatened by thousand cankers disease. Systemic imidacloprid and dinotefuran applications were made to mature black walnut trees to evaluate their translocation and concentration levels in various tissue types including leaf, twig, trunk core, nutmeat, and walnut husk. The metabolism of imidacloprid in plants produces a metabolite, olefin-imidacloprid, which has been documented to have insecticidal properties in other systems. Trunk CoreTect (imidacloprid) soil pellets and a trunk spray of dinotefuran were applied to mature black walnuts in spring 2011. Imidacloprid concentrations were detected in both the lower and upper strata in all tissue types tested and progressively increased through month 12 post-treatment in twig and leaf tissue. Olefin-imidacloprid was detected in the nutmeat and walnut husk. Dinotefuran was only detected in the first sampling period and was found in low concentration levels in leaf and twig tissue types, and was not detected in the trunk, nutmeat or the walnut husk.

Understanding the imidacloprid metabolism in long-term exposure through a comparative study of imidacloprid and its major metabolite levels in the urine and hair of intentionally exposed rabbits

Understanding the imidacloprid metabolism in long-term exposure through a comparative study of imidacloprid and its major metabolite levels in the urine and hair of intentionally exposed rabbits

Identification of imidacloprid metabolites in onion (Allium cepa L.) using high‐resolution mass spectrometry and accurate mass tools

Imidacloprid is a potent and widely used insecticide on vegetable crops, such as onion (Allium cepa L.). Because of possible toxicity to beneficial insects, imidacloprid and several metabolites have raised safety concerns for pollenating insects, such as honey bees. Thus, imidacloprid metabolites continue to be an important subject for new methods that better understand its dissipation and fate in plants, such as onions. One month after a single addition of imidacloprid to soil containing onion plants, imidacloprid and its metabolites were extracted from pulverized onion with a methanol/water-buffer mixture and analyzed by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOF-MS) using a labeled imidacloprid internal standard and tandem mass spectrometric (MS/MS) analysis. Accurate mass tools were developed and applied to detect seven new metabolites of imidacloprid with the goal to better understand its fate in onion. The accurate mass tools include: database searching, diagnostic ions, chlorine mass filters, Mass Profiler software, and manual use of metabolic analogy. The new metabolites discovered included an amine reduction product (m/z 226.0854), and its methylated analogue (m/z 240.1010), and five other metabolites, all of unknown toxicity to insects. The accurate mass tools were combined with LC/QTOF-MS and were able to detect both known and new metabolites of imidacloprid using fragmentation studies of both parent and labeled standards. New metabolites and their structures were inferred from these MS/MS studies with accurate mass, which makes it possible to better understand imidacloprid metabolism in onion as well as new metabolite targets for toxicity studies.

Aldehyde Oxidase Importance In Vivo in Xenobiotic Metabolism: Imidacloprid Nitroreduction in Mice

Aldehyde oxidase (AOX) metabolizes many xenobiotics in vitro, but its importance in vivo is usually unknown relative to cytochrome P450s (CYPs) and other detoxification systems. Currently, the most important insecticides are neonicotinoids, which are metabolized in vitro by AOX on reduction of the nitroimino group and by CYPs via oxidation reactions. The goal of this study was to establish the relative importance of AOX and CYPs in vivo using the mouse model. The procedure was to reduce liver AOX activity by providing tungsten or hydralazine in the drinking water or to use the AOX-deficient DBA/2 mouse strain. None of these approaches reduced CYP activity measured in vitro with an isozyme nonspecific substrate. Liver AOX activity was reduced by 45% with tungsten and 61% with hydralazine and 81% in AOX-deficient mice relative to controls. When mice were treated ip with the major neonicotinoid imidacloprid (IMI), metabolism by CYP oxidation reactions was not appreciably affected, whereas the AOX-generated nitrosoguanidine metabolite was decreased by 30% with tungsten and 56% with hydralazine and 86% in the AOX-deficient mice. The other IMI nitroreduction metabolite, desnitro-IMI, was decreased by 55%, 65%, and 81% with tungsten, hydralazine, and in the AOX-deficient mice, respectively. Thus, decreasing liver AOX activity by three quite different procedures gave a corresponding decrease for in vivo reductive metabolites in the liver of IMI-treated mice. Possible AOX involvement in IMI metabolism in insects was evaluated using AOX-expressing and AOX-deficient Drosophila, but no differences were found in IMI nitroreduction or sensitivity between the two strains. This is the first study to establish the in vivo relevance of AOX in neonicotinoid metabolism in mammals and one of the first for xenobiotics in general.