Worldwide use of neonicotinoids continues to expand as pest populations develop resistance to the once-widely-used pesticide classes including the organophosphates and methylcarbamates. The most commonly used neonicotinoids are IMI and TMX, the primary focus of these studies. The overall goal is to further understand the metabolism of neonicotinoids relative to: in vivo importance of AOX, metabolic pathways of the novel neonicotinoid, cycloxaprid and mechanisms of TMX hepatotoxicity and hepatocarcinogenicity. CYPs have been shown to be involved in in vitro and in vivo neonicotinoid metabolism, but the relative in vivo importance of AOX is unknown. AOX is implicated to play a role in the nitroreduction of N-nitroguanidine neonicotinoids, the most prominent subclass. There is considerable variability in the activity of AOX between species and individuals which may be reflected in differences in neonicotinoid metabolism and detoxification. Secondly, CYC is a new neonicotinoid that is under development to control IMI-resistant pests. However, its metabolic pathway has yet to be determined, particularly in reverting to its potent nAChR agonist precursor, nitromethylene-imidazole. Finally, TMX is the only neonicotinoid to produce liver toxicity and tumors in chronically-treated mice, but not rats. Earlier studies concluded that formation of dm TMX and iNOS inhibition by dm-CLO is likely the mechanism of TMX toxicity. Furthermore, differences in metabolic rates between species may explain the mouse specific toxicity. However, the molecular mechanism of TMX or dm-TMX hepatotoxicity/ hepatocarcinogenicity remains unclear. It is critical to fully understand the metabolic/ enzymatic pathways of neonicotinoids and mechanisms of toxicity as their use continues to increase and for future pesticide design.The nitro substituent on neonicotinoids is important relative to their potency and selectivity for the insect nAChR. From the seven commercial neonicotinoids, approximately 100 metabolites have been identified in plants and mammals, some of which are bio-activated and can interact with the mammalian nAChR.
A number of studies have demonstrated the importance of CYPs in neonicotinoid metabolism in vitro and in vivo. However,macetas plastico cuadradas the role of AOX in neonicotinoid metabolism has yet to be established in vivo, especially in the oxidative- and CYP-rich environment of the liver. AOX is important in xenobiotic metabolism. This enzyme is expressed mainly in liver but is also present in many other tissues with variations in activity depending on species, gender, age, drug usage and disease states. Tungsten or hydralazine in the diet or drinking water results in reduced AOX activity in guinea pigs, rabbits and mice. There are even notable differences in AOX activity between strains of mice , e.g. compared to CD-1 mice, the DBA/2 strain is deficient in the expression of AOX homologue 1 and homologue 2 and has reduced expression of AOX1. Since AOH1 and AOX1 are the primary AOX genes expressed in mouse liver , DBA/2 mice are an appropriate AOX-deficient model for studies on in vivo mammalian xenobiotic metabolism. The wide range of inter- and intra-species AOX activity may result in different rates of neonicotinoid metabolism and detoxification in mammals and insects. Despite the increasing significance of AOX, there have been very few studies examining the in vivo contribution of this enzyme to xenobiotic metabolism. Mice can serve as a surrogate for humans since AOX activity in IMI nitroreduction in vitro is comparable between these two species. This study uses chemical inhibitors and genetic deficiency for mice and Drosophila melanogaster to evaluate the relevance of AOX in neonicotinoid metabolism in vivo. Mouse liver cytosol and microsomes were prepared by homogenizing liver in ice-cold PBS using a Sonic Dismembrator followed by centrifugation of the homogenate at 1,000g for 10 min and then the supernatant at 10,000g for 30 min. An aliquot of the 10,000g supernatant was recovered for AOX activity analysis and the remainder was centrifuged at 100,000g for 1 h to collect the CYP-containing microsomal pellet fraction which was resuspended in PBS for protein measurement and the CYP activity assay.
Mouse liver cytosol was added to 50 µM DMAC solution and the reaction monitored by an absorbance decrease using a VersaMax microplate reader at 398 nm for 5 min with an average control value of -18.4 mOD/min. 7-Ethoxycoumarin is a broad-specificity substrate used to measure the activity of many CYP enzymes by monitoring the oxidation to 7-hydroxycoumarin. Microsomes were mixed with 50 mM 7- ethoxycoumarin in assay buffer glycerol and 0.1 mM EDTA and prewarmed at 37°C for 5 min. After addition of 10 mM NADPH , reactions were incubated at 37°C for 30 min in a shaking water bath.Samples were extracted with chloroform , briefly vortexed, then centrifuged at 3,000g for 5 min. The organic phase was removed and added to 30 mM sodium borate and vortexed. Following centrifugation at 3,000g for 5 min, the upper layer was recovered and plated on a Costar 96-well black plate and fluorescence read at an excitation wavelength of 370 nm and an emission wavelength of 460 nm using a SpectraMax M2 Microplate Reader with an average control value of 11.2 nmol 7-hydroxycoumarin/mg protein.AOX is a potentially important factor in drug metabolism with many studies examining its in vitro inhibition and the proposed effects on xenobiotic action. There is a wide range of AOX activity between species with rabbits, monkeys and humans the highest, mice intermediate and rats and dogs having the lowest activity. This same species dependent relationship is also observed for in vitro IMI nitroreduction by liver cytosol. Tungsten and hydralazine treatments provide a way to reduce AOX activity in vivo in mammals to evaluate its relevance in xenobiotic metabolism. Tungsten replaces molybdenum at the active center of AOX, rendering it inactive , but the mechanism of AOX inactivation by hydralazine is unknown.The level of AOX inhibition by tungsten treatment in mice was less than that by hydralazine , a difference reflected in their effect on IMI metabolism. Hydralazine treatment resulted in significantly reduced IMI metabolism to IMI-NNO and IMI-NH,maceta redonda but tungsten treatment only significantly reduced IMI metabolism to IMI-NH. There are four AOX genes in mice with two of the variants being expressed in the liver, AOH1 and AOX1. DBA/2 mice are completely deficient in the expression of AOH1 and have low expression of AOX1 compared to CD-1 mice. Our data also establish that DBA/2 mice have significantly lower AOX activity in the liver and further show that the reduced AOX activity decreased IMI metabolism to IMI-NNO and IMI-NH, but not to IMI-5-OH or IMI-ole.
The AOX-generated IMI metabolites are not all detoxification products. IMI-NH is a likely contributor to the nicotinic effects of IMI. It is over 300 times more potent than IMI at the mammalian nAChR and the mouse ip toxicity is also increased several fold. IMI-NNO retains insecticidal activity and as an N-nitroso compound it was subjected to extensive toxicological tests and cleared of potential problems. Our study concludes that reduced AOX activity is tightly correlated with reduced IMI metabolism to IMI-NNO and IMI-NH indicating that these products are mostly from AOX, not CYPs. Based on the metabolic sequence and relevant correlations, IMI-NH is mostly formed via IMI-NNO rather than another pathway. This is the first report on the metabolism of CYC with an emphasis on in vivo metabolites in mice at 15 and 120 min post-treatment. Metabolites are not conclusively identified since synthetic standards were not available. However, based on calculated m/z values and characteristic chlorine isotope patterns, it is concluded that CYC is converted in part to NMI, but is mostly oxidized to multiple mono- and dihydroxylation products within 15 min and dissipate by 120 min in liver. There are many possible isomeric CYC oxidation products from hydroxylation on the 6-, 7-, 10- or 11- position in each case with two possible stereoisomers of which five are detected as distinct peaks by LC/MS. Minor products include 2-CYC and nitroreduction on the nitro group to NO-CYC and NH2-CYC. CYC is readily hydrolyzed to NMI. Therefore, extraction and analytical conditions were carefully chosen to limit degradation. However, minor amounts of NMI detected may be due to degradation of CYC rather than its in vivo metabolism. NMI per se was not extensively metabolized. Only minor amounts of one hydroxylation and one nitroso product were evident. Detection methods may limit observing the extent of metabolism since only 3 mg/kg was administered to mice. The findings reported here lay the background for future studies on characterization of metabolites and in vitro species comparisons. In vitro conditions will allow detection of more metabolites and the enzymes responsible for their formation can be determined.
TMX undergoes metabolic activation to CLO in insects, plants and mice. The neonicotinoids generally have favorable mammalian toxicology with the exception of TMX which is a hepatotoxicant and hepatocarcinogen in mice but not rats or dogs. The mechanism of this TMX- and mouse-specific hepatotoxicity/ hepatocarcinogenicity is of considerable interest relative to neonicotinoid risk assessment. Green et al.observed that liver microsomal metabolic rates are greater for mouse than rat or human in the production of TMX metabolites, dm-TMX, CLO and dm-CLO. They also found that mouse-specific adverse effects of TMX are due to dm-TMX exacerbated by dm-CLO which mimics the structure of L-NAME, a standard inhibitor of iNOS, an enzyme with a regulatory role in the development of hepatotoxicity. The structure-activity relationships of neonicotinoids as hepatotoxicants or hepatocarcinogens help focus mechanistic hypotheses on specific molecular substituents. TMX and dm-TMX are hepatotoxicants/ hepatocarcinogens and contain the oxadiazinane substituent uniquely among the neonicotinoids so this moiety is of particular interest. N-Methyl substituents on five of the neonicotinoids are not the hepatotoxic moiety because dm-TMX lacks this group. The Green et al.hypothesis is that dm-TMX is the hepatotoxicant exacerbated by dm-CLO as an iNOS inhibitor. The present study examines an alternative hypothesis that the unique aspect of the oxadiazinane moiety is its metabolic conversion to HCHO and N-methylol intermediates , the ultimate hepatotoxicants and hepatocarcinogens which may be synergized by dm-CLO as a NOS inhibitor.high-throughput oxymyoglobin assay by following the procedure of Dawson and Knowles. To determine nNOS inhibition, HEK293 cells overexpressing rat nNOS were cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin and 0.4 mg/mL geneticin. For enzyme activity assays, cells were cultured in Costar 96-well black plates and treated with 5 µM A23187 ionophore to activate nNOS then with dm-CLO or L-NAME for 8 h. To measure nitric oxide production, cell media was removed and replaced with reaction buffer containing 1 mM L-arginine, 10 µM 4,5-diaminofluorescein diacetate , 1 mM NADPH and dm-CLO or L-NAME. After a 2-h incubation in the dark, fluorescence was read at an excitation wavelength of 490 nm and an emission wavelength of 520 nm using a SpectraMax M2 Microplate Reader.Mouse liver microsomes were prepared by homogenizing livers from male albino Swiss Webster mice in PBS followed by differential centrifugation of the supernatant. The microsomal 100,000g pellet was resuspended in PBS and protein concentration measured. Liver microsomes for species comparison studies were from BD Biosciences. Recombinant CYP3A4 was compared to recombinant CYP2C19 , the two isoforms previously shown to be responsible for TMX metabolism. Each neonicotinoid was incubated with microsomes from mouse, rat or human or rCYP isoform and 0 or 1 mM NADPH in PBS for 1 h at 37°C. Alachlor and hexamethylphosphoramide , compounds known to produce HCHO via N-methylol intermediates on activation by CYPs , were also incubated under the same conditions with mouse liver microsomes alone or with NADPH. Enzymatic reactions were terminated by addition of 25% ZnSO4 aqueous solution and saturated Ba2 aqueous solution. Samples were briefly vortexed and placed on ice for 5 min then centrifuged at 18,000g for 5 min. HCHO levels were analyzed after conversion to the 2,4-dinitrophenylhydrazine derivative. An aliquot of the supernatant was mixed with 7.2 mM DNPH and incubated at room temperature for 30 min followed by addition of carbon tetrachloride , vortexing for 30 seconds and a final incubation at room temperature for 30 min. The lower organic layer was evaporated to dryness under N2 at 25°C and resuspended in 80:20:0.1 ACN/water/HCO2H and filtered through 0.2 µm nylon for HPLC analysis. Samples were analyzed on a Waters Alliance 2695 HPLC equipped with an Agilent Zorbax SB-C18 column and Waters Alliance 2487 dual UV absorbance detector.