Toxicity Evaluation And Pharmacological Property Analysis Of Neonicotinoid Insecticides And Their Analogues

Neonicotinoid insecticides have brought a revolution to agricultural production on the advent of pest prevention and control.Especially following the emergence of imidacloprid,it has played key roles in improving crop yields.In the modern agricultural production,the chemical control is still the main way among the three major means of pest control.However,because of the long term using and even abusing of neonicotinoid insecticides and the other chemical pesticides,some pests have developed high resistance to imidacloprid and the cross-resistance to other neonicotinoid insecticides,which has become new problems.Therefore,the development of new chemical insecticides for the alternate use is particularly demanded.The nAChRs are neonicotinoid insecticides target.By comparing the structure of a series of new neonicotinoid insecticides,the trans-conjugate structure of nitro（-N02）or cyano（-CN）groups have been found to be the functional groups of insect nAChR selective agonists,such as imidacloprid and dinotefuran.Recently,with the nitro and cyano on cis-position,a series of compounds with insecticidal activities have been reconstructed.It is proved that this method can be a potential way to develop new insecticides.This research is mainly on the toxicity evaluation and pharmacological property analysis of neonicotinoid insecticides and their analogues.1.Bioassay of imidacloprid,cycloxaprid,paichongding（PCD）and their enantiomersThe toxicities of imidacloprid,cycloxaprid,PCD and their enantiomers on Nilaparvata lugens（the brown planthopper,BPH）had been determinated.The results showed that imidacloprid,cycloxaprid and cycloxaprid-P2 had the highest insecticidal activity,with LD₅₀ of 0.153,0.163 and 0.199 ng/insect respectively.In contrast,cycloxaprid enantiomer P1(LD₅₀=1.122 ng/insect)showed much lower activity than that of cycloxaprid and cycloxaprid-P2.Two PCD enantiomers,PCD-P1（9.72 ng/insect）and PCD-P2（9.13 ng/insect）,showed significantly lower acitivities than PCD and its enantiomers PCD-P3 and PCD-P4,with LD₅₀ values of 5.122,2.801 and 2.642 ng/insect respectively.The results showed that imidacloprid,cycloxaprid and cycloxaprid-P2 have relatively high activities.2.The pharmalogical properties of PCD and its enantiomersThe potencies of PCD and its enantiomers were analyzed and compared with imidacloprid using electrophysiological experiments.In Xenopus oocytes expression system,the nAChRs Nlα1/β2 and Nlα1_Y151S/β2 were expressed respectively.The evoked-currents of nAChRs activated by imidacloprid,PCD and its analogues were recorded and analyzed.The results showed that,on both receptors,imidacloprid had higher potencies than that of PCD and its enantiomers,reflecting in the bigger I_max values and less EC₅₀ values.When comparing PCD and its enantiomers,PCD-P3 and PCD-P4 showed higher potencies than PCD and the other two enantiomers,PCD-P1 and PCD-P2.The results clear showed that the receptors were more sensitive to PCD-P3 and PCD-P4,which will be more toxic to insects.3.The effects of neonicotinoids and their analogues on the detoxification anzyme activitivesThis section mainly focused on the detoxification enzymes activities in BPH when treated with imidacloprid and its analogue IPP165059,or cycloxaprid and its analogue IPP165030.BPH were treated with the doses of LD₁₅ and LD₅₀,and then survival insects were collected to determine the detoxification anzyme activities,mainly including cytochrome P450 monooxygenase,carboxylesterase and glutathione transferase.In 24 h,the results for P450 monooxygenases showed that the LD₅₀ doses of all tested compounds could significantly increase the enzyme activitites,to 3.3-3.9-fold of control.In the LD₁₅ doses,only IPP 165030 caused an obvious increase in enzyme activity（4.59-fold）.In 48 h,the increases in P450 enzyme activitives were only detected in the treatments with IPP165059 and IPP165030 at LD₁₅ doses（1.69-fold and 1.74-fold）.The results for carboxylesterase（CarEs）activitives showed that the treatment with IPP165030 at LD₁₅ dose in 24 h had the greatest influence on enzyme activitives（up to 4.5-fold）.However,all compounds have little influence on CarEs of BPH in 48 h.The results for glutathione transferase（GST）activitives showed that the LD₁₅ doses of IPP165030 induced GSTs activitives to 4.52-fold in 24 h.The LD₅₀ doses of cycloxaprid and IPP165030 also induced GST activitives to 2.06-and 2.39-fold.Taken together,these results demonstrated that insecticides at low doses could induce the enzyme activities more significantly than that at medium doses in 24 h.4.The effect of insecticides and their analogues on mRNA levels of detoxification anzyme genesThe influences of imidacloprid at LD₅₀ dose on mRNA levels of detoxification anzyme genes were determined by qRT-PCR.The expression levels of all P450 monooxygenase genes,categorized into Clade2,Clade3,Clade4 and Mito families,from BPH were evaluated.Eight genes in Clade4 family were over-expressed,in which CYP4CE1 CYP417A1 and CYP425B1 were increased to 3.35-fold,3.94-fold and 3.12-fold of that of control.In Clade3 family,seven genes were up-regulated significantly,including CYP6CS1,CYP6CW1 and CYP6ER1（up to 5.63,4.91 and 4.42-fold）.However,in Mito and Clade2 families,only two genes were small but still significantly up-regulated,and one gene（CYP303A1）was significantly down-regulated to 2.94-fold.In 24 h after the insecticide treatment,cycloxaprid at LD₁₅,LD₅₀ and LD85 doses and IPP165030 at LD₅₀ dose could up-regulate the expression of one CarE gene,to 1.6-1.9-fold.Interestingly,the expression of this CarE gene was down-regulated in 48 h following the insecticide treatment with imidacloprid and IPP165059 at LD₁₅ and LD₅₀ doses.