Effects Of Azocyclotin On Endocrine Disruption And Its Mechanism In Xenopus Laevis

Azocyclotin, a widely used oganictin insecticide, has rarely been reported about the negative effects of toxicity on aquatic animals. In this study, X. laevis was used as targeted animal for the evaluation of acute toxicity in larvae and tadpoles, exposure to azocyclotin caused malformation in larvae, thyroid endocrine disruption in tadpoles, relative genes of hypothalamus-pituitary-gonad endocrine disruption and sexual development in froglets. The main results of these experiments are as follows:The acute toxicity showed that the 96 h-LC50 value of azocyclotin to embryos and tadpoles were 1.84μg/L and 0.786 μg/L, and the calculated environmental safety concentrations were 0.02 μg/L and 0.008 μg/L respectively. These results suggested that azocyclotin was highly toxicity to X. laevis, meanwhile, embryos were more sensitive than tadpoles.The results of Frog Embryo Terato genesis Assay-Xenopus (FETAX) after 96 h exposure to azocyclotin demonstrated that the 96 h-EC50 value of azocyclotin to embryos was 1.329 μg/L. The teratogenic index was 1.41 means that azocyclotin has the teratogenic effect on X. laevis. Concurrently, the weight, sarcomere width, fin width of larvae were decreased and notochord deformation, caudal vertebra deformation, pericardial edem, facial deformity were observed after exposure. The narrow fins was the specific malformation type of oganictin. Azocyclotin caused triiodothyronine (T3) levels decreasing and tetraiodothyronine (T4) levels increasing dramatically, by which the disorder of thyroid hormone levels might lead to malformation because of unbalanced development of the tissues and organs in X. laevis embryos we speculated. The mRNA expressions of genes involved in teratogenicity about apoptosis was observed. Casepase3 (cpp32β) up-regulated and caspase 1(ice) down-regulated significantly means that azocyclotin might interfere the apoptosis pathway signed by Fas protein. To be specific, the improvement of activated Cpp32 lead to inactivation of Ice, and then Cpp32 execute the apoptosis program and promote the apoptosis of some cells, eventually leading to abnormal occurrence. Up-regulation of organizer-specific secreted-dorsalizing factor{chordin) and down-regulation of sex determining region Y-box 9 (sox9), matrix metalloproteinase 2{mmp2) were observed, which suggested that azocyclotin had certain effects to induce malformation in X. laevis skeletal or notochord development.In the Amphibian Metamorphosis Assay (AMA) study, stage 51 X. laevis tadpoles were exposed to different concentrations of azocyclotin (0,0.02,0.1 and 0.5 μg/L) for 21 days, during which time the majority stage of tadpole were undergoing metamorphosis. Exposure to azocyclotin caused an inhibition effects on metamorphie development of X. laevis, including shorten hind limb length and reduced the developmental rate of stage. Azocyclotin caused alterations in the T3 contents, indicating thyroid endocrine disruption Real-time PCR was performed to examine the expression levels of genes involved in TH signaling pathway. Significantly down-regulation of type 2 deiodinase gene was observed, which may partially responsible for the decreased T3 concentrations. Furthermore, the expression of T3 response genes, including thyroid hormone receptor (tr/3), basic transcription element binding protein (bteb), stwmelysin-3(st3) and mmp2 were down-regulated in tadpoles, suggesting azocyclotin caused the decrease of T3 contents and in turn affect the mRNA expression of downstream genes involved in multiple physiological response.In the exposure lasting six months, from the beginning of the embryo to three months after the completion of metamorphosis. The results demonstrated that azocyclotin brought about reducing of survival rate, body length, body weight and delayed the metamorphosis completed, also the number of female pheno type dec lined and hermaphrodite individuals increased compared to the corresponding control group. In females, a significant decrease in LSI, GSLand the transcriptional levels of cyp19 gene were observed, while the gene expression of ar increased dramatically in both males and females. These results suggested that azocyclotin might inhibit testosterone conversion to estradiol through interfereing the synthesis of aromatase, which leads to accumulation of testosterone in the X. laevis froglets, and promote gene type female X. laevis develop into male phenotype. Consequantely, azocyclotin might exhibit certain androgenie effect.In conclusion, azocyclotin had the effect on teratogenesis, interfered metamorphosis process by TH-dependent signaling pathway regulating, and affected sexual development in froglets at the environmental exposure concentration.