Molecular Toxicity And Ecotoxicology Effects On Chinese Toad (Bufo Bufo Gargarizans) Exposed To Several Pesticides

Pesticides are necessary and non-substituted production material a quite long time in future.People have an attention to the negative effect of using pesticides largely with increasing deterioration of human health and entironment.Especially, there is a growing concern about assessment healthy and ecological risk of pesticides in worldwide,which were discussed by the expert and governmental officeholder.Ecotoxicology is science foundation of hazard assessment of pesticides.Studying of representative organism individual and colony are the basic method of ecotoxicology.Amphibian is a very important role in environmental safety assessment. Laboratory experiments have provided basic data about the effects of pesticides on model organisms——Xenopus laevis,but ultimately we would like to know pesticides affect a diversity of amphibian or a wildlife amphibian under natural condition. Furthermore,the relative lack of target specificity has raised concems about their potential to cause adverse effects on non-target wildlife populations.Chinese toad,Bufo bufo gargarizans,is frequently found in rice field,muddy ponds,wetlands and other aquatic ecosystems in southern area of China.In the present paper,we studied the impact of exposure to insecticides(chlorpyrifos, diazinon,fipronil) and herbicides(butachlor,acetochlor,paraquat,chlorimuron-ethyl, quinclorac),which are extensively and plentifully used in rice or cereal fields,on Chinese toad in laboratory.The content included general toxicity effect(acute toxicity, subchronic toxicity),chemical molecular mutation(gene mutation,chromosome aberration),reproductive toxicity and embryotoxicity.We also have gone deep into the mechanism of mutation,and chemical carcinogenesis,including DNA strand breaks, DNA-Protein cross-links,RAPD,DNA methylation and cell apoptosis.The results showed that: (1) General toxicity effects:the results indicated that the acute lethality of butachlor and chlorpyfifos on Bufo bufo gargarizans tadpoles was very high. Acetochlor,diazinon and paraquat were moderately toxic,while fipronil,and chlofimuron-ethyl was relatively low.Continuously,chlorpyrifos and acetochlor leaded to the potential subchronic toxicity risk to Bufo bufo gargarizans tadpoles.(2) Chemical mutation:The first evident result was significant(P<0.01) induction of MN in Bufo bufo gargarizans tadpole specimens due to exposure of high concentrations of chlorpyrifos,butachlor,diazinon,acetochlor and paraquat than the control group except fipronil,quinclorac and chlorimuron-ethyl.It indicated they induced chromosomal lesion of erythrocytes of Bufo bufo gargarizans tadpoles. Further,Significant positive correlations(correlation coffecient(r) at 5%level) were found between the concentrations of chlorpyrifos,diazinon,fipronil,acetochlor and paraquat and DNA damage measured by OTM in Comet assay,except quinclorac and chlorimuron-ethyl.To sum up,they could cause the genotoxic effects in erythrocytes of Bufo bufo gargarizans tadpoles by MN and comet assay.(3) Reproductive toxicity and embryotoxicity:chlorpyrifos,diazinon,butachlor, acetochlor and paraquat increased percentage of asbnormal shaped sperm cells in Bufo bufo gargarizans.Besides,total hatch was greatly affected in embryos exposed to chlorpyrifos,diazinon,fipronil,acetochlor and paraquat.But the values of TI less than 1.5,they could not cause malformation of Bufo bufo gargarizans.Based on experimental evidence obtained,the grouping adult Bufo bufo gargarizans were exposed to chlorpyrifos,diazinon,butachlor,acetochlor and paraquat for 10 d by hyodermic.As follows:(1) DNA strand breaks assay:the results of the investigation indicated that the above pesticides result in DNA strand breaks of the liver cells and erythrocytes in the adult Bufo bufo gargarizans by Comet assay.(2) DNA-Protein cross-links were measured by modification of the comet assay. Since exposure of slides to proteinase K can increase DNA migration of the treated cells,the presence of DPC can be indicated by compare of the tail moment before and after the proteinase K added.The results suggested that the above pesticides could cause DPC of erythrocytes in male.(3) DNA base mutation assay:the random amplified polymorphic DNA(RAPD) assay has been shown to detect genotoxin-induced DNA damage and mutations.The present study,we evaluated the possible target in genomic DNA of liver cells of male that exposed to the above pesticides.Two 10-mer RAPD primers(S₁ and S₈) produced the most distinguishable banding profiles between pesticides-treated and control genomic DNA.(4) DNA methylation evaluation:DNA methylation plays an important role in the regulation of gene expression in eukaryotes.Effect of chlorpyrifos,diazinon, butachlor,acetochlor and paraquat on DNA methylation level is studied.High performance liquid chromatography(HPLC) is used to determine the content of DNA bases.It is found that DNA methylation levels in hepatocytes treated with chlorpyrifos,diazinon,butachlor,acetochlor and paraquat are significantly lower than that by control.The results indicate that the above pesticides can decrease DNA methylation levels.(5) Cell apoptosis studies:we investigated the above pesticides could induce apoptosis in erythrocytes by Giemsa-Wright dying and Comet assay.The results indicated that the pesticides induced an increase of erythrocytes apoptosis index in a dose-dependent manner,and induced DNA fragmentation in liver cells determined by agarose gel electrophoresis.In conclusion,the present study explores the ecotoxicology and mutation effects of insecticides and herbicides on Chinese toad(Bufo bufo gargarizans),reveals the toxic mechanism of insecticides and herbicides on Chinese toad,and provides a scientific method for pesticides pollution monitoring and scientific data for the ecological assessment risk of pesticides.