Teratogenic Effects And Mechanism Of Chlopyrifos And Acetochlor Combined Exposure On Embryos Of Xenopus Tropicalis

Chlopyrifos is an important insecticide in our country, Acetochlor, a member of the chloroacetanilide family of herbicides, is widely used. Some studies have showed, the rapid decline of amphibian was closely related to the use of pesticides. So in this study we chose these two pesticides exposing to Xenopus tropicalis embryos using the acute toxicity test, frog embryo teratogenicity test(FETAX), tissue sections experiments, real-time PCR to elucidate the teratogenic effects and mechanism of chlopyrifos and acetochlor single exposure and combined exposure to embryos of Xenopus tropicalis.First, we detected the value of TC50 after embryos were exposed to chlopyrifos and acetochlor in separate group and combined group for 96 h. When embryos were exposed to chlopyrifos and acetochlor separately, the TC50 was 0.4327mg/L and 2.70 mg/L respectively.When chlopyrifos combined with 2.70 mg/L acetochlor, the TC50 was 0.3493 mg/L while the number was 1.06 mg/L when acetochlor combined with 0.4327 mg/L chlopyrifos. In conclusion,chlopyrifos and acetochlor are two kinds of typical teratogens. The study also shows that combined exposure leads to higher percentage of teratogenic toxicity than single exposure and there was a dose-response relationship between the proportion of malformations and Pollution Concentration.In addition, primary malformation in both single and combined exposure test is seen in pericardial edema, swollen cloaca, bent notochord, curved tail. According to the study, embryos in combined group were more likely to suffer these malformations. Furthermore, there was a clear dose-response relationship between the proportion of suffering malformation of curved tail and single chlopyrifos pollutant concentration with a upward trend. Likewise, similar dose-response relationships were observed between malformation of bent notochord and curved tail and single Acetochlor pollutant concentration, malformation of pericardial edema as well as bent notochord and combined pollutants concentration of chlopyrifos and acetochlor(2.70 mg/L), malformation of curved tail and combined pollutants concentration of chlopyrifos(0.4327mg/L) and acetochlor.Moreover, tissue sections experiments of embryos,which gave a more detailed explanation about the histological mechanism of malformation confirmed these previous observations added with a new type of malformation in eyes.In the last stage, real-time PCR method was used in detecting differences of HOXA3 、HOXA11 、 HOXA13 in gene expression levels. In the group with combined pollutants of chlopyrifos and acetochlor(2.70 mg/L) exposing to embryos for 96 h, the result presented a dose-response relationship between gene expression levels of HOXA11、HOXA13 as well as the malformation of bent notochord and pollutants concentration. This relationship also can be seen inthe group with combined chlopyrifos(0.4327mg/L) and acetochlor pollutants lasted for 96 h between gene expression levels of HOXA3 、 HOXA11 、 HOXA13 as well as malformation of curved tail and the concentration of pollutants. On the other hand, in the groups exposed to single doses of chlopyrifos and acetochlor lasted for both 48 h and 96 h, the dose-response relationship did not exist between gene expression levels and malformation. In this way, we can draw a conclusion that gene expression differences of HOXA11、HOXA13 are related to malformations of bent notochord and curved tail. Likewise, there is a relationship between gene expression differences of HOXA11 and cloaca. However, the relationship between gene expression differences of HOXA3 and eye malformation still need to be tested and verified.