| Acetochlor is a chloracetamide herbicide, inhibiting protein synthesis in weed, which is one of the three most widely used herbicides in China. Since it has the qualities of better water-soluble and relatively low soil sorption, it is easy to shift from farming to the shallow groundwater through the infiltration or with rainwater runoff into surface water. Acetochlor have been detected frequently in the environment and classified by the US EPA as a B-2 carcinogen. Thus, it is important to investigate the toxic effects of acetochlor for ecological risk assessment. Zebrafish (Danio rerio) is a premiere model organism widely used for the screening and risk assessment of endocrine disruption chemicals (EDCs). The present study evaluated the effects of herbicide acetochlor on endocrine system of zebrafish as well as the potential mechanisms, including hypothalamus-pituitary-thyroid axis (HPT), hypothalamic-pituitary-gonadal axis (HPG) and antioxidant enzyme systems.A method for the simultaneous detection of acetochlor and 16 hormones in fish and water samples was developed by a modified QuEChERS-SPE-UHPLC-MS/MS. All of the parameters involved in QuEChERS extraction and the SPE clean-up step, such as the SPE type, eluting solvent and pH, have been optimized to achieve maximum recoveries and minimum matrix effects. Percentage recoveries for fish ranged from 73.0 to 118.8 and for water from 70.3 to 117.1. The detection limits for fish ranged from 0.03 to 9.04 ng/g and for water from 0.01 to 0.98 ng/mL. Excellent linearity, precision, accuracy and satisfactory recoveries were obtained in the present study, which can provide a technical support for further exploration of the effects of acetochlor on steroidogenesis in zebrafish.The results of adult fish acute toxicity test indicated that LC50 (96 h) values of acetochlor were 2.90 mg/L, which were moderately toxic and detected in gonad with no enrichment. The results of embryo acute toxicity test indicated that LC50 (120 h) values of acetochlor were 5.09 mg/L, which were less sensitive than adults. Exposed to various acetochlor from 6 to 120 h post-fertilization (hpf) causing the decreased survival rate and hatching rate of zebrafish embryos and increased malformation rate, including malformations of body bent and pericardial edema..The potential effects of window exposure to acetochlor on thyroid system of zebrafish were assessed in the present study. Zebrafish larvae (7 d post-fertilization, dpf) were exposed to a series concentration of acetochlor for 14 days window up until 21 dpf. The high concentrations of acetochlor (30,100 and 300 μg/L) resulted in decreasing T3, increasing T4 levels significantly and weight gain. The mRNA expressions of genes involved in thyroid hormone synthesis (tshβ, slc5a5 and tpo) and thyroid hormone receptor (trα) were up-regulated significantly in zebrafish larvae exposed to acetochlor, while the genes related to thyroid hormone metabolism (dio1 and ugt1ab) and transport (ttr) were down-regulated significantly. The results were similar to those of positive control T3 exposure, which indicated that acetochlor exhibited the growth promoting effect as T3. From above all, it was induced that acetochlor might be competitively bound to thyroid receptor (TR), causing the interference T4/T3 balance in the body and then inducing outgrowing of zebrafish larvae. The excessive secretion of thyroid stimulating hormone (TSH) accelerated the synthesis of T4/T3 by increasing the feedback of hypothalamic and pituitary, which might result in the adverse effect on the function of the thyroid gland.The effects of short-term exposure to acetochlor on gonad system of zebrafish were also evaluated. After the 14-day exposure to acetochlor (0,0.03 and 0.3 mg/L), there were no significant differences on gonadosomatic index (GSI) and liver somatic index (LSI) compared with the control. Acetochlor was detected in zebrafish ovaries and increased in a time-dependent manner during exposure. Acetochlor exposure significately increased 17-OHP levels and decreased the levels of T, E2 and ASD. Meanwhile, acetochlor exposure significately up-regulated the mRNA expressions of era, cyp17a1, vtg Ⅰ, vtg Ⅱ and down-regulated the mRNA expressions of ar, apgr, cyp19a, cyp19b, 17bhsd1 and 17bhsd3. The results of the changes of hormones and transcriptional level were similar to those of positive control E2 exposure. It was further explained that acetochlor might reduce the conversion rate of ASD to T and E2 through inhibiting of the activity of 17β-HSD and CYP19, and meanwhile competitively inhibit androgen receptors (AR) with T. Thus, acetochlor had potential anti-androgen effects or estrogen-like effects.Furthermore, we evaluated the effect of acetochlor on antioxidant enzyme system and energy metabolic enzymes. After the 14-day exposure to acetochlor (0,0.03 and 0.3 mg/L), the activities of SOD, CAT, POD, GST and the levels of GSH increased firstly and then decreased with the increase of stress levels; The levels of MDA increased with the increase of stress levels; The activities of ATP, Na+K+-ATP and Ca2+Mg2+-ATP were inhibited with the increase of stress levels. In the liver, a large number of free radicals might be produced in metabolic processes of acetochlor. The activities of antioxidant enzyme system including SOD, CAT, POD and GST were stimulated with the low concentration of acetochlor. However, the activities of antioxidant enzyme system reached their peak and could not work as normal with the increase of exposure time and concentration, when the free radicals began to attack such as amino acids, enzymes, enzyme activity and resulted in reduction of enzyme activity. On the other hand, a large number of free radicals could cause the increase of lipid peroxidation effect and lead to accelerate the content of MDA, which further inhibit the activities of ATP, Na+K+-ATP and Ca2+Mg2+-ATP.In the study, the disrupting effects of acetochlor on HPT, HPG and the antioxidant enzyme system of zebrafish were evaluated systematically, and the possible mechanisms of its endocrine disruption were summarized. Results revealed that acetochlor in the environment could affect both fish growth and reproductive endocrine through the interference of the key enzymes of thyroid hormones, steroid hormones and the receptors. Acetochlor could potentially affect the aquatic ecosystem and human health. Therefore, the use of the pesticide needs strict regulation. |
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