Developmental toxicity assessment of neonicotinoids and organophosphate esters with a human embryonic stem cell-and metabolism-based fast-screening model

In recent years,neonicotinoids(NEOs)and organophosphate esters(OPEs)have been widely used as substitutes for traditional pesticides and brominated fame-retardants,respectively.Previous studies have shown that those compounds can be frequently detected in environmental and human samples,are able to penetrate the placental barrier,and are toxic to animals.Thus,it is reasonable to speculate that NEOs and OPEs may have potential adverse effects in humans,especially during development.We employed a human embryonic stem cell differentiation-and liver S9 fraction metabolism-based fast screening model to assess the potential embryonic toxicity of those two types of chemicals.We show that four NEO and five OPE prototypes targeted mostly ectoderm specification,as neural ectoderm and neural crest genes were down-regulated,and surface ectoderm and placode markers up-regulated.Human liver S9 fraction's treatment could generally reduce the effects of the chemicals,except in a few specific instances,indicating the liver may detoxify NEOs and OPEs.Our findings suggest that NEOs and OPEs interfere with human early embryonic development.

Synthesis,insecticidal activity and stability study of novel nitromethylene neonicotinoids with five-membered aromatic heterocycles

Twenty-four novel neonicotinoid analogues with nitromethylene and five-membered aromatic heterocycles were designed and synthesized.All target molecular structures have been confirmed by analytical and spectral data.Some compounds exhibited notable insecticidal activities against aphid(Aphis medicaginis)and brown planthopper(Nilaparvata lugens).The aqueous stability test confirmed that the stabilities of those compounds were superior to the leading compound,and the photostability was even better than that of imidacloprid.

Water bridges are essential to neonicotinoids: Insights from synthesis,bioassay and molecular modelling studies

Water-bridged H-bonds have been observed in many cases of ligand-receptor recognitions. To explore the roles of water bridges in the binding of neonicotinoids with receptors, twenty-four neonicotinoid compounds with nine fragments, including 1 H-1,2,3-triazole, CN, COOMe, CONHNH_2, CONHMe, NO_2,NH_2, NHCOMe and NHCSNH_2 were synthesized and evaluated, of which, compounds with cyano group showed the best activities against Aphis craccivora. Accordingly, the cyano group is the optimal fragment mimicking the water bridge. Two cyano-substituted cis-nitromethylene compounds display good insecticidal activities, whereas the LC_(50) values are lower than those of their corresponding prototypes.Docking study showed that the cyano group acts only as H-bond acceptor, while the water bridge can act as both donor and acceptor. It revealed that the water bridge might be stable in the active site and was not suitable to be replaced by other groups. The findings illustrated that the water bridge is necessary for high insecticidal activities of neonicotinoids, which should be also helpful in better understanding the binding mode of neonicotinoids.

Computational Investigations about the Effects of Heteromolecular Aggregation on Bioactivities:a Case of Neonicotinoids and Water

Molecular aggregation state of bioactive compounds plays a key role in bio-interactive procedure.Diverse ag-gregation states of bioactive compounds contribute to different biological or chemical properties.Water-bridge,as the simple hetero-molecular aggregation,has been found bridging the binding between many bioactive compounds and their targets through hydrogen bonding network,e.g.in the recognition of neonicotinoids with insect nAChRs.To better understanding the roles of water-bridge on bioactivities of compounds,an approach of hetero-dimeric ag-gregation with water was proposed.Quantitative structure-activity relationship(QSAR)and pharmacophore mod-eling investigations were applied on 19 neonicotinoids,as well as their aggregates with water.The aggregate-based CoMSIA,PHASE and linear QSAR models presented better statistical significance and predictabilities than the monomer ones,which indicated that the bioactivities correlated with the aggregate properties and water bridged hy-drogen bond of the active site.All results revealed the essential roles of water-bridge in ligand recognition,which should be considered in future ligand design and optimization.

Association between new onset type 1 diabetes and real-world antibiotics and neonicotinoids’exposure-related gut microbiota perturbation

Background The real-world exposure levels of non-therapeutic antibiotics and neonicotinoids in type 1 diabetes(T1D)children and their associations as environmental triggers through gut microbiota shifts remained unknown.We thus investigated the antibiotics and neonicotinoids’exposure levels and their associations with gut microbiota in pediatric T1D.Methods Fifty-one newly onset T1D children along with 67 age-matched healthy controls were recruited.Urine concentrations of 28 antibiotics and 12 neonicotinoids were measured by mass spectrometry.Children were grouped according to the kinds of antibiotics’and neonicotinoids’exposures,respectively.The 16S rRNA of fecal gut microbiota was sequenced,and the correlation with urine antibiotics and neonicotinoids’concentrations was analyzed.Results The overall detection rates of antibiotics were 72.5%and 61.2%among T1D and healthy children,whereas the neonicotinoids detection rates were 70.6%and 52.2%(P=0.044).Children exposed to one kind of antibiotic or two or more kinds of neonicotinoids had higher risk of T1D,with the odd ratios of 2.579 and 3.911.Furthermore,co-exposure to antibiotics and neonicotinoids was associated with T1D,with the odd ratio of 4.924.Antibiotics or neonicotinoids exposure did not affect overall richness and diversity of gut microbiota.However,children who were exposed to neither antibiotics nor neonicotinoids had higher abundance of Lachnospiraceae than children who were exposed to antibiotics and neonicotinoids alone or together.Conclusion High antibiotics and neonicotinoids exposures were found in T1D children,and they were associated with changes in gut microbiota featured with lower abundance of butyrate-producing genera,which might increase the risk of T1D.

Synthesis and insecticidal activities of novel bridged-neonicotinoids

A series of novel bridged-neonicotinoid analogues were designed and synthesized, which were constructed by starting material 8 with cyclopentenone or cyclohexenone in the presence of catalyst aluminium chloride. All of the compounds were characterized and confirmed by^1H NMR,^（13）C NMR, HRMS and IR. The bioassay tests showed that compounds 5 and 6a showed higher bioactivities than imidacloprid against Aphis craccivora.

Effect of pesticides on phosphorylation of tau protein,and its influence on Alzheimer’s disease

Alzheimer’s disease(AD)is a progressive and neurodegenerative illness which results in alterations in cognitive development.It is characterized by loss/dysfunction of cholinergic neurons,and formation of amyloid plaques,and formation of neurofibrillary tangles,among other changes,due to hyperphosphorylation of tau-protein.Exposure to pesticides in humans occurs frequently due to contact with contaminated food,water,or particles.Organochlorines,organophosphates,carbamates,pyrethroids and neonicotinoids are associated with the most diagnosed incidents of severe cognitive impairment.The aim of this study was to determine the effects of these pesticides on the phosphorylation of tau protein,and its cognitive implications in the development of AD.It was found that exposure to pesticides increased the phosphorylation of tau protein at sites Ser198,Ser199,Ser202,Thr205,Ser396 and Ser404.Contact with these chemicals altered the enzymatic activities of cyclin-dependent kinase 5 and glycogen synthase kinase 3 beta,and protein phosphatase-2A.Moreover,it altered the expression of the microtubule associated protein tau gene,and changed levels of intracellular calcium.These changes affected tau protein phosphorylation and neuroinflammation,and also increased oxidative stress.In addition,the exposed subjects had poor level of performance in tests that involved evaluation of novelty,as test on verbal,non-verbal,spatial memory,attention,and problem-solving skills.

Characterisation of Neonicotinoid and Pymetrozine Resistance in Strains of Bemisia tabaci(Hemiptera:Aleyrodidae)from China

Four strains of the Q biotype and one of the B biotype of the whitefly Bemisia tabaci collected from China were characterised for resistance to four neonicotinoid insecticides and pymetrozine.Q biotype strains showed moderate to strong resistance to imidacloprid,thiamethoxam and acetamiprid,but little or no cross-resistance to dinotefuron.Resistance to neonicotinoids was consistently associated with resistance to pymetrozine,despite the latter having a distinct（though unresolved）mode of action.The single B biotype strain proved largely susceptible to all the insecticides investigated.Resistance in the Q biotype strains was associated with over-expression of a cytochrome P450 monooxygenase gene,CYP6CM1,whose substrate specificity presumably accounts for the observed cross-resistance profiles.

Design and Synthesis of 3-(1-Phenylethyl)thiazolidine-2-thione Derivatives with Potential Biological Activities

A series of novel neonicotinoids derivatives 4a―4p was designed and synthesized by introducing the ethylbenzene group into the thiazole ring, their structures were confirmed by IR, 1H NMR and elemental analysis. The crystal structure of compound 4j has been determined by X-ray diffraction analysis.

S-(+)-(E)-1-(2-furfuryl)-5-substituted-1,3,5-hexahydrotriazine-2-N-nitroimines:Synthesis,Crystal Structure and DFT Calculations

A novel neonicotinoid analogue（C13H19N5O5,3a）had been synthesized,the structure was characterized by elemental analysis,IR and 1H NMR spectra,and the S-（＋）-（E）-configuration was confirmed by single-crystal X-ray diffraction.The crystal belongs to monoclinic,space group P21 with a = 8.7076（17）,b = 8.3211（17）,c = 10.642（2）,β = 92.370（3）o,V = 770.4（3）3,Z = 2,Dc = 1.402 g/cm3,μ = 0.110 mm-1,Mr = 325.33,F（000）= 344,S = 1.027,R = 0.0543 and wR = 0.1229 for 3601 unique reflections with 2919 observed ones（I 2σ（I））.Compound 3a is stabilized by intramolecular hydrogen bonds and intermolecular force.In addition,the structure of compound 3a was optimized by the B3LYP/6-31G（d,p）.DFT/B3LYP optimizations were performed based on X-ray geometries applying 6-31G（d,p）basis set.The optimized structure of compound 3a by the B3LYP/6-31G（d,p）method is more bent than in the crystal.IR spectrum of the solid compound is consistent with the X-ray structure.The HOMO-LUMO gap in 3a（5.3 eV）indicates high kinetic stabilities of compound 3a.The preliminary bioassay test showed that 3a exhibited good activities against Nilaparvata legen,Pseudaletia separate Walker and Aphis medicagini at 500 mg/L.

Synthesis, Crystal Structure and Insecticidal Activities of Novel Neonicotinoid Derivatives

Ten new N-oxalyl derivatives of neonicotinoid compound were designed and synthesized. Their structures were confirmed by ^1H NMR spectra, elemental analysis and X-ray diffraction. The preliminary insecticidal activities of the new compounds were evaluated. The results of bioassays indicate that the title Compounds exhibit moderate insecticidal activities. Surprisingly, the insecticidal activity of compound 7b against bean aphids at 200 mg/kg is 100%, which is comoarable to that of the commercialized imidacloprid.

Synthesis and Crystal Structure of the Optical Activity of (Z)-Nitromethylene Neonicotinoid Analogue

A novel （Z）-nitromethylene neonicotinoid analogue （C23H27Cl2N5O4·2H2O） （II） with optical activity has been synthesized, the structure was characterized by elemental analysis, IR and 1H NMR spectra, and the （Z）-configuration was confirmed by single-crystal X-ray diffraction. The crystal belongs to triclinic, space group P1 with a = 7.4638（3）, b = 12.6232（5）, c = 15.2990（6）, α = 71.907（1）, β = 89.397（2）, γ = 80.314（1）°, V = 1349.28（9）3, Z = 2, Dc = 1.340 g/cm3, μ = 0.286 mm-1, Mr = 544.43, F（000） = 572, S = 1.056, R = 0.0801 and wR = 0.2366 for 4998 unique reflections with 3012 observed ones （I 〉 2σ（I））. In the crystal, the dihedral angle between the pyridine and 4-Cl-phenyl rings is 58.13°. Intermolecular O-H···O, C-H···O and C-H···Cl hydrogen bonds involving water molecules stabilize the crystal structure.

Aldehyde oxidase mediated enantioselective metabolic health risk of dinotefuran

Chiral pollutants often pose significant differential environmental health risks.In this study,the biotransformation of chiral dinotefuran(DIN)and its enantioselective metabolic toxicity mechanisms have been systemically investigated.Firstly,reversedphase chromatography-high resolution mass spectrometry was developed to quantify the content of DIN R/S chiral enantiomer with pg level sensitivity,revealing a lower elimination rate constant(K_(e))of S-DIN(0.730 h^(-1))than R-DIN(0.746 h^(-1)).Secondly,the interaction mechanism between DIN metabolism and important endogenous bioactive molecules,such as aldehyde oxidase(AOX)and neurotransmitters,was revealed.The DIN nitro-group was converted into a guanidine group by the reducing site of nearby flavin adenine dinucleotide(FAD)in AOX with the preferred higher affinity of S-configuration.Meanwhile,the endogenous tryptophan(Trp)aldehyde metabolic intermediate,5-hydroxyindoleacetaldehyde(5-HIAL),provides a persistent electron donor for DIN reduction via the oxidation-catalyzed site in AOX,resulting in remarkable up-regulation of monoamine neurotransmitters such as serotonin and dopamine.Thirdly,the higher level of neurotransmitters further mediated dysregulation of oxylipin homeostasis via the serotonergic pathway,where S-DIN exhibited more pronounced liver lipid damage and environmental health risk with the accumulated lipid biomarkers,oxidized triglyceride(OxTG)and oxidized sphingomyelin(OxSM).This study elucidates the AOX-mediated enantioselectivity metabolic pathway of DIN,providing a new analytical method for chiral pollutants and paves the way for their health risk assessments.

Expression profile of CYP402C1 and its role in resistance to imidacloprid in the whitefly, Bemisia tabaci

Bemisia tabaci(Gennadius)(Hemiptera:Aleyrodidae)is a cosmopolitan insect pest causing serious damage to crop production.Cytochromes P450(CYPs)of B.tabaci are widely known to be involved in the metabolic resistance to a variety of insecticides,continuously increasing the difficulty in controlling this pest.In this study,four P450 genes(CYP6CMI,CYP6CXl,CYP6CX3,and CYP402CI)in B.tabaci exhibited correlations with the resistance to imidacloprid.We have focused on trying to understand the function and metabolism capacity of CYP402C1.The expression profiles of CYP402C1 were examined by reverse transcription quantitative real-time PCR and fluorescence in situ hybridizations.Its role in resistance to imidacloprid was investigated by RNA interference,transgenic Drosophila melanogaster,and heterologous expression.The results showed that CYP402CI was highly expressed in the active feeding stages of B.tabaci,such as nymphs and female adults.CYP402C1 was mainly expressed in midguts of nymphs and adults,especially in the filter chamber.Knockdown of CYP402CI significantly decreased the resistance of B.tabaci to imidacloprid by 3.96-fold(50%lethal concentration:186.46 versus 47.08 mg/L).Overexpression of CYP402CI in a transgenic D.melanogaster line(Gal4>UAS-CYP402CI)significantly increased the resistance to imidacloprid from 12.68-to 14.92-fold(129.01 and 151.80 mg/L versus 1925.14 mg/L).The heterologous expression of CYP402CI showed a metabolism ability of imidacloprid(imidacloprid decreased by 12.51%within 2 h).This study provides new insights for CYP402C1 function in B.tabaci and will help develop new strategies in B.tabaci control and its insecticide resistance.

Adsorption behavior of imidacloprid pesticide on polar microplastics under environmental conditions:critical role of photo-aging

The photo-aging behavior of microplastics(MPs)in natural environment has become a global concern.The ultraviolet radiation has enough energy to change the polymer structure and physicalchemical properties of MPs.Less attention has focused on the interactions of the photo-aged polar and biodegradable MPs with organic pollutants.This work investigated the structural properties of aged polar polyamide(PA)MPs and biodegradable polylactic acid(PLA)MPs exposed to ultraviolet irradiation and their adsorption behavior and mechanism for neonicotinoid insecticide imidacloprid(IMI).The results showed that the MPs had extensive changes in surface morphology and chemical properties after photo-aging.The C–N bond of PA MPs was disrupted to form more carbonyl groups.The oxygen-containing functional groups on the surface of aged PLA MPs were broken and generated relatively smaller molecules.The adsorption capacity of IMI on PA MPs decreased by 19.2%,while the adsorption capacity of IMI on PLA MPs increased by 41.2%after photo-aging.This depended on the natural structure of the MPs and their ability to absorb ultraviolet light.The electrostatic interactions,hydrogen bonds,van der Waals interactions,and polar-polar interactions were the main adsorption mechanisms of IMI on MPs.High initial solution pH and low ionic strength favored the adsorption of IMI by altering charge distribution on the MPs surface.The formation of the humic acid-IMI complexes decreased the concentration of IMI in the water phase and further decreased the adsorption on MPs.These results are enlightening for a scientific comprehension of the environmental behavior of the polar MPs.

Insights into Free and Conjugated Forms of Neonicotinoid Insecticides in Human Serum and Their Association with Oxidative Stress

Following exposure,neonicotinoid insecticides(NEOs)can be metabolized by both Phase Ⅰ and Phase Ⅱ reactions catalyzed by human cytochrome P450 enzymes.However,toxicities of parent NEOs and their metabolites are still unclear,and little is known about biotransformation rates and pathways of NEOs in humans.In this study,98 serum samples collected in China were analyzed for free,conjugated and total forms of six parent NEOs(i.e.,acetamiprid(ACE),imidacloprid(IMI),clothianidin(CLO),thiacloprid(THD),thiamethoxam(THM),and dinotefuran(DIN))and four metabolites(i.e.,N-desmethyl-acetamiprid(N-dm-ACE),1-methyl-3-(tetrahydro-3-furylmethyl)(DIN-U),5-hydroxy-imidacloprid(5-OH-IMI),olefin-imidacloprid(Of-IMI)).NEOs and their metabolites were detected in all serum samples,and the total median concentrations of free,conjugated,and total forms of 10 NEOs were 2.04,2.01,and 5.12 ng/mL,respectively.Conjugated forms of NEOs accounted for only half(53%)of the total forms of NEOs.Based on the profiles of Phase Ⅰ and Phase Ⅱ metabolites of NEOs in serum,it was found that age is a determinant in Phase Ⅰmetabolism of DIN and Phase Ⅱ metabolism of IMI.The Phase Ⅱmetabolites of NEOs are associated with oxidative DNA damage,and the conjugated forms of IMI,DIN,and 5-OH-IMI in serum were significantly positively correlated with oxidative stress.Overall,the amount of NEOs present in conjugated forms in human serum was determined to document the existence of a considerable proportion of free forms of these insecticides.

Assessment of the Bemisia tabaci C YP6CM l vQ transcript and protein levels in laboratory and field-derived imidacloprid-resistant insects and cross-metabolism potential of the recombinant enzyme

Over-expression of the cytochrome P450 CYP6CM1 gene has been associated with imidacloprid resistance in a number of Q and B biotype Bemisia tabaci laboratory strains from distinct geographical origins worldwide. We recently demonstrated that the Q biotype version of the CYP6CM 1 protein （CYP6CMlvQ） is capable of metabolizing imida- cloprid. Here, we show that the levels of BtCYP6CMlvQ were also elevated in laboratory- resistant strains and field-derived populations, with variable imidacloprid resistance levels, collected in Crete. High levels of CYP6CMlvQ transcripts were also determined in survivors of a heterogeneous field population, after exposure to discriminating imidacloprid dosage. Using peptide antibody-based detection assays, we demonstrated that in line with transcriptional data, the CYP6CMlvQ protein levels were higher in imidacloprid-resistant insects, which further implicates the gene as the causal factor of resistance. Finally, assess- ment of the cross-metabolism potential of CYP6CMlvQ against additional neonicotinoid molecules used for B. tabaci control revealed that clothianidin and thiacloprid, but not acetamiprid or thiamethoxam, are metabolized by the recombinant enzyme in vitro.

Development of an analytical method based on solid-phase extraction and LC-MS/MS for the monitoring of current-use pesticides and their metabolites in human urine

Pyrethroids,organophosphorus pesticides and fipronil have been listed as priority chemicals in human biomonitoring studies because of their wide use and potential health effects in humans.The determination of 13 pesticides,including pyrethroids(deltamethrin,cypermethrin,permethrin,cyfluthrin,bifenthrin),organophosphorus(chlorpyrifos,chlorpyrifosmethyl,and malathion),fipronil,neonicotinoids(imidacloprid,acetamiprid and thiacloprid)and triazole(prothioconazole),together with 13 corresponding metabolites in human urine samples was achieved by solid-phase extraction and analysis by liquid chromatography coupled to tandem mass spectrometry.All targeted compounds,except malathion dicarboxylic acid,were measured with a mean within-accuracy(n=5)of 71%–114%(RSD:1%–14%)and between-run(n=15)accuracy of 80%–118%(RSD:2%–14%).Limits of quantitation of the targeted analytes ranged from 0.1 to 16 pg/m L.The detection result of urine samples from 25 volunteers indicated that the detection frequencies of 3,5,6-trichloro-2-pyridinol(median:448 pg/m L),6-chloropyridine-3-carboxylic acid(median:193 pg/m L),2-methyl-3-phenylbenzoic acid(median:181 pg/m L),3-phenoxybenzoic acid(median:99 pg/m L),2-isopropyl-6-methyl-4-pyrimidinol(median:77 pg/m L),cyfluthrin(median:59 pg/m L),cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid(cis-DCCA,median:53 pg/m L),trans-DCCA(median:25 pg/m L),prothioconazole(median:21 pg/m L),imidacloprid(median:7 pg/m L),and prothioconazole-desthio(median:1 pg/m L)were>50%.The obtained results show that the validated method is suitable for the human biomonitoring of these currentuse pesticides and their metabolites.

Facile three-component synthesis and insecticidal evaluation of hexahydroimidazo[1,2-a]pyridine derivatives

A series of new hexahydroimidazo[1,2-a]pyridine derivatives were synthesized via convenient and practical three-component reactions. Preliminary bioassays showed that majority of the target compounds exhibited moderate to excellent insecticidal activity against cowpea aphids （Aphis craccivora）. Among them, compound 91 demonstrated significant activity with LCso value of 0.00918 mmol/L which was about 3.8-fold higher than that of imidacloprid （IMI）. Furthermore, the study of stereostructure-activity relationship of four isomers of 9k indicated that configuration played a key role in insecticidal activity of these compounds.

Comparative study of the effect of solvents on the efficacy of neonicotinoid insecticides against malaria vector populations across Africa

Background:New insecticides with a novel mode of action such as neonicotinoids have recently been recommended for public health by WHO.Resistance monitoring of such novel insecticides requires a robust protocol to monitor the development of resistance in natural populations.In this study,we comparatively used three different solvents to assess the susceptibility of malaria vectors to neonicotinoids across Africa.Methods:Mosquitoes were collected from May to July 2021 from three agricultural settings in Cameroon(Njombe-Penja,Nkolondom,and Mangoum),the Democratic Republic of Congo(Ndjili-Brasserie),Ghana(Obuasi),and Uganda(Mayuge).Using the CDC bottle test,we compared the effect of three different solvents(ethanol,acetone,MERO)on the efficacy of neonicotinoids againstAnopheles gambiae s.l.In addition,TaqMan assays were used to genotype key pyrethroid-resistant markers inAn.gambiae and odds ratio based on Fisher exact test were used to evaluate potential cross-resistance between pyrethroids and clothianidin.Results:Lower mortality was observed when using absolute ethanol or acetone alone as solvent for clothianidin(11.4-51.9% mortality in Nkolondom,31.7-48.2% in Mangoum,34.6-56.1% in Mayuge,39.4-45.6% in Obuasi,83.7-89.3% in Congo and 71.1-95.9% in Njombe pendja)compared to acetone+MERO for which 100% mortality were observed for all the populations.Similar observations were done for imidacloprid and acetamiprid.Synergist assays(PBO,DEM and DEF)with clothianidin revealed a significant increase of mortality suggesting that metabolic resistance mechanisms are contributing to the reduced susceptibility.A negative association was observed between the L1014F-kdr mutation and clothianidin resistance with a greater frequency of homozygote resistant mosquitoes among the dead than among survivors(OR=0.5;P=0.02).However,the I114T-GSTe2 was in contrast significantly associated with a greater ability to survive clothianidin with a higher frequency of homozygote resistant among survivors than other genotypes(OR=2.10;P=0.013).Conclusions:This study revealed a contrasted susceptibility pattern depending on the solvents with ethanol/acetone resulting to lower mortality,thus possibly overestimating resistance,whereas the MERO consistently showed a greater efficacy of neonicotinoids but it could prevent to detect early resistance development.Therefore,we recommend monitoring the susceptibility using both acetone alone and acetone+MERO(4μg/ml for clothianidin)to capture the accurate resistance profile of the mosquito populations.