Endocrine Disruping Effects Of Butachlor On Zebrafish(Danio Rerio)

Butachlor is a chloracetamide herbicide, which is one of the top three herbicides for controlling weeds in rice fields in China. Zebrafish (Danio rerio) is a model organism widely used for evaluating reproductive toxicity and endocrine-disrupting effects of chemicals due to its characters of small size, ease of culture, short life cycle and prolific egg production rate with high rate of fertilization and hatching. The present study evaluated the effects of butachlor on endocrine system of zebrafish were evaluated and the potential mechanisms.The result of acute toxicity test on adult zebrafish showed that the LC50value of96%butuchlor within96h was0.49mg/L, indicating that butachlor was highly toxic to zabrafish. The normal levels of thyroxine (T4) and3,5,3’-triiodothyronine (T3) during the development in zebrafish were also measured. The result showed that whole-body content of both T4and T3remained stable during the pre-hatch period (0-3d). After hatching, both T3and T4significantly increased, with peaking at10days post-fertilization (dpf) and21dpf, respectively, and then thyroid hormones subsequently declined in later development. This work has provided the baseline data for future studies of the thyroid system in zebrafish.The potential effects of butachlor on hypothalamus-pituitary-thyroid (HPT) axis of zebrafish were assessed in the present study. The parameters to be measured include body weight, length, and the contents of T4and T3. In order to assess the potential mechanisms of thyroid disruption of butachlor, the expressions of related genes in HPT axis were determined. The result demonstrated that butachlor could increase the condition factor of zebrafish in the early life stage after30d’s exposure, while no difference of condition factor of zebrafish was observed between the control group and treated group in adult stage; T4and T3levels in zebrafish were increased after exposure to butachlor in both the early life stage and adult stage. After the exposure of butachlor, the mRNA levels of the genes of corticotrophin-releasing hormone (CRH), thyroid-stimulating hormone (TSH), transthyretin (TTR), deiodinase (Deiol) and thyroid hormone receptor (TRa) and Slc5a5were significantly upregulated in zebrafish of the early life stage, while the mRNA level of uridinediphosphate-glucuronosyl transferase (UGTlab) was significantly downregulated. No significant differences were observed in expressions of the genes thyroid peroxidase (TPO), globulin (TG), D2and TRβ. In summay, the hypothalamus-pituitary-thyroid (HPT) axis of zebrafish was affected by butachlor.The effects of butachlor on hypothalamus-pituitary-gonad (HPG) axis of zebrafish were also evaluated. The fecundity was assessed during the exposure period in a short-term assay. At the end of the exposure period, various endpoints such as gonadosomatic index (GSI), liver somatic index (LSI), and the levels of plasma sex steroids (T and E2) and vitellogenin (VTG) of zebrafish were examined. In order to assess the potential mechanisms of reproductive disruption of butachlor, the expressions of related genes in hypothalamus-pituitary-gonad (HPG) axis were determined in the early life stage of zebrafish. Our result showed that hypothalamus-pituitary-gonad (HPG) axis of zebrafish was affected by butachlor:(1) The number of eggs and number of spawning events per female were significately reduced after exposure to butachlor;(2) The gonadosomatic index (GSI) in males was decrased, while no affect on liver somatic index (LSI) was observed in both females and males;(3) The levels of T and E2in plasma were significately decreased in females, and the levels of vitellogenin (VTG) in males were significately increased.(4) There was no significant alteration in expression of vitellogenin (VTGI), estrogen receptor (ERα, ERβ1, ERβ2) and aromatase (CYP19a) in the early life stage after the exposure of butachlor, whereas the transcription of VTG II gene was significantly upregulated.Addtionally, the liver of zebrafish, which is the organ responsible for detoxifying exogenous substance, was used to evaluate the effect of butachlor on antioxidant system of the fish. Our results showed that either the activitie of hepatic SOD and GST, or the contents of GSH in female zebrafish were significantly decreased, while the activity of CAT was not altered.