Teratogenic Effect And DNA Damage On Early Stages Of Development Of Xenopus Tropicalis Exposed To Three Acetanilide Herbicides

Worldwide amphibian declines have been a concern for biologists for the past several decades. The causes of such declines may include habitat loss, invasive species, pathogens, and man-made chemicals. Pesticides, in particular, were known to interfere with reproduction in amphibians and were likely contributing to population declines. Pesticides used widely in agricultural caused pollution of ecological environment while bring great economic benefits for human. Based on this background, we chose three acetanilide herbicides (acetochlor, pretilachlor, butachlor) used widely in China as object, studied the teratogenic effect and DNA damage on early stages of development of Xenopus tropicalis.First, acute toxicity of three herbicides were determinated on two model organisms (X. laevis and X. tropicalis). The results showed that three herbicides were moderately toxic to two kinds of tadpoles, and the toxicity level were butachlor> acetochlor> pretilachlor, X. tropicalis was relatively sensitive to three agents. Therefore, we chose X. tropicalis as object for the study in this paper.Next, using FETAX test, tissue section and whole mount in situ hybridization technique to study the teratogenic effect of three herbicides on early stages of development of X.tropicalis. Results of FETAX test showed that embryonic median lethal concentration of herbicides were 2.26 mg/L,4.67 mg/L and 1.06 mg/L, respectively; median teratogenic concentrations were 1.2 mg/L,3.84 mg/L and 0.63 mg/L, respectively; TI values were 1.88,1.22 and 1.68, which suggest that acetochlor and butachlor had potentially teratogenic risk. Additionally, three herbicides could induce a variety of malformations on X.tropicalis embryos, such as abdominal swelling, facial deformities, curvature of the spine, tail bent and head deformities. To further confirm whether the herbicides had an impact on the development of cartilage, the heads of the abnormal individuals were observed by histological examination. The results showed that development of cartilage of head showed significant difference, and resulted in smaller ventricles, compared with the control group. Then the Sox9 gene related with cartilage development and Dmrt5 gene related with brain development were detected expression pattern in embryo exposed to butachlor, using whole mount in situ hybridization technique. The results showed that the expression of Sox9 gene was imbalance, Dmrt5 expression was inhibited. Therefore, Sox9 and Dmrt5 gene were candidate target genes of butachlor, which could provide favorable information for the mechanism.The teratogenic effect on embryos, but also the DNA damage was studied on tadpoles. After the tadpoles of X. tropicalis on 47 stages were exposed to sub-lethal concentrations of three herbicides for 4 days, the erythrocytes were test by micronucleus test and Comet assay. The micronucleus frequency of blood cells and nuclear anomalies in the high concentration group was significantly higher, indicating three herbicides can cause chromosome damage; Comet assay results showed that the DNA damage indicators with acetochlor, pretilachlor, butachlor concentration increased and showed a significant linear correlation. We could draw the following conclusions:Three herbicides could change the genetic material of tadpoles, cause DNA damage. The results also provided scientific basis for screening suitable biomarkers.