Elsevier

Science of The Total Environment

Volume 653, 25 February 2019, Pages 1140-1148
Science of The Total Environment

Enantioselective thyroid disruption in zebrafish embryo-larvae via exposure to environmental concentrations of the chloroacetamide herbicide acetochlor

https://doi.org/10.1016/j.scitotenv.2018.11.037Get rights and content

Highlights

  • (+)-S-acetochlor had stronger thyroid disruptive effect in zebrafish larva.

  • Acetochlor exposure increased T3 contents and decreased T4 contents.

  • (+)-S-acetochlor could induce developmental toxicity at 2 μg/L.

Abstract

Acetochlor (ACT) is a chiral chloroacetamide pesticide that has been heavily used around the world, resulting in its residues being frequently found in surface waters. It has been reported that ACT is an endocrine disrupting chemical (EDC) with strong thyroid hormone-disrupting activity in aquatic organisms. However, the enantioselectivity underlying thyroid disruption has yet to be understood. In this study, using a zebrafish embryo-larvae model, the enantioselective thyroid disruption of ACT was investigated at a series of environmentally relevant concentrations (1, 2, 10 and 50 μg/L). Our results showed that both racemic ACT and its enantiomers significantly increased the malformation rates of embryos at 72 h postfertilization (hpf). Decreased thyroxine (T4) contents and increased triiodothyronine (T3) contents were found in larvae at 120 hpf, with (+)-S-ACT exhibiting a greater effect than (−)-R-enantiomer. Similarly, (+)-S-ACT also showed a stronger effect on the mRNA expressions of thyroid hormone receptors (TRα and TRβ), deiodinase2 (Dio2) and thyroid-stimulating hormone-β (TSHβ) genes. The observed enantioselectivity in TR expressions was consistent with that of in silico binding analysis, which suggested that (+)-S-enantiomer binds more potently to the TRs than (−)-R-enantiomer. In general, ACT enantiomers showed different influences on the secretion of THs, expression of TH-related key genes and binding affinity to TRs. Considering the different toxicity of different enantiomers, our study highlights the importance of enantioselectivity in understanding of thyroid disruption effects of chiral pesticides.

Introduction

The extensive use of pesticides represents a constant threat to humans and wildlife because of the unwanted potential of interfering with biological systems. An increasing number of pesticides are endocrine-disrupting chemicals (EDCs) (McKinlay et al., 2008; Jiang et al., 2016), some of which are suspected to have thyroid-disrupting effects (Picard-Aitken et al., 2007; Tu et al., 2016a). Acetochlor (ACT), which is among the most heavily used herbicides worldwide, belongs to the chloroacetamide herbicide family. The annual consumption of ACT in China is up to 107 kg (Ye, 2003). Because of the large application amounts, ACT residues have been ubiquitously found in aquatic systems (Fernandez-Cornejo et al., 2014; Yuan et al., 2018). In the United States, ACT is the third most frequently detected herbicide in natural waters, with concentrations of up to 2.5 μg/L in Midwest streams (Foley et al., 2008; Scribner et al., 2000). Furthermore, in paddy fields, the concentration of ACT is up to 30.9 μM (Ma et al., 2000; Xu et al., 2016). The widespread existence of ACT is posing a great threat to the eco-environment. The U.S. EPA has classified ACT as a B-2 carcinogen (USEPA, 2007). Many studies have also demonstrated that ACT is a thyroid-disrupting chemical (Crump et al., 2002; Helbing et al., 2006; Li et al., 2009a; Jiang et al., 2015; Yang et al., 2016;). For example, by accelerating T3-dependent metamorphosis, ACT is able to affect the developmental processes of amphibians (Crump et al., 2002). In addition, exposure to ACT could also result in tissue-specific alternative expression of thyroid hormone-related genes in fish (Li et al., 2009a).

Thyroid hormones (THs) play a vital role in the endocrine system, mediating the growth, development and metabolism of vertebrates (Power et al., 2001; Tu et al., 2016b). Thyroid homeostasis results from a multiloop feedback system, which is controlled by the hypothalamus-pituitary-thyroid (HPT) axis. For example, the hypothalamus secretes thyrotropin-releasing hormone (TRH) and induces the pituitary to release thyroid-stimulating hormone (TSH), which regulates TH synthesis and release (Carr and Patino, 2011; Chen et al., 2018; Moreira et al., 2018). Due to the complex regulatory network of thyroid hormones, multiple targets may be affected by exposure to EDCs. Many previous studies have indicated that the expression of TH receptors and deiodinase genes is a sensitive molecular biomarker for thyroid disruption in fish exposed to environmental contaminants (Picard-Aitken et al., 2007; Xiang et al., 2017).

Unlike many other herbicides, most of the chloroacetamides are chiral compounds. An increasing number of studies have shown that chiral pesticides often exhibit significant enantioselectivity in biological activities and environmental behaviors (Ye et al., 2009; Sun et al., 2011). Understanding and utilizing enantioselectivity are viewed as important approaches for accurate risk assessment of chiral pollutants. To date, there have been several studies regarding the chiral separation and enantioselective environmental behaviors of chloroacetamides (Cai et al., 2011; Liu et al., 2017; Maronić et al., 2018). However, to the best of our knowledge, few studies have considered the effect of enantioselectivity on aquatic toxicity at the environmentally relevant levels. In particular, the overall knowledge of the enantioselective effects of chiral compounds on the disruption of thyroid system is rather limited.

In this study, we employed ACT as a representative chiral chloroacetamide and zebrafish as a vertebrate model. The aquatic toxicities of racemic ACT ((±)-rac-ACT) and its two enantiomers ((+)-S and (−)-R) were compared. The primary objective of this study was to explore the role of enantioselectivity of ACT in the thyroid disruption of zebrafish embryo-larvae and the associated mechanisms. The difference caused by different enantiomers of ACT in developmental toxicity, THs contents and mRNA expression levels of HPT-related genes was elucidated. In addition, molecular docking was applied to better describe the enantioselective interactions of ACT with thyroid hormone receptor (TR) proteins.

Section snippets

Chemicals and enantiomer preparation

Acetochlor (>98.5%, racemic) was purchased from Sigma-Aldrich (St. Louis, Missouri, USA). The enantiomers of ACT were separated using a Jasco LC-2000 series HPLC system (Jasco, Tokyo, Japan) equipped with a PU-2089 quaternary gradient pump, a mobile phase vacuum degasser, an AS-1559 autosampler with a 100-μL loop, a CO2060 temperature-controlled column compartment, a UV-2075 plus ultraviolet (UV)/visible detector and a variable-wavelength CD-2095 circular dichroism (CD) detector. The employed

Separation and identification of enantiomers

The enantiomers of ACT were baseline resolved under the optimized chromatographic conditions (Fig. 1). The chiral HPLC analysis showed that the purities were 99.6% and 99.8% for the isolated (−)-R-ACT and (+)-S-ACT, respectively. The good separation and purity ensured that the individual enantiomers could be further used in the subsequent experiments to evaluate the enantioselective effect of ACT on aquatic toxicity.

Concentrations of ACT and its enantiomers in exposure water

The analysis of water samples showed that the measured concentrations of ACT in

Discussion

The objective of the present study was to elucidate the enantioselective effects of chiral ACT on the thyroid system in zebrafish embryo-larvae. Based on exposure experiments to zebrafish with the racemate and the two enantiomers of ACT at environmentally relevant concentrations, we found that during the early development of zebrafish, the enantioselectivity of chiral ACT existed in the developmental abnormalities, expression of HPT axis genes and thyroid homeostasis.

Conclusions

In summary, we found that the racemate and the two enantiomers of ACT induced developmental toxicity and altered the whole body T4 and T3 levels and the mRNA expressions of genes involved in the HPT axis in zebrafish via different mechanisms. The present study suggests that (+)-S-ACT had stronger disruption to the thyroid and could result in more significant hypothyroidism of zebrafish. These findings highlight the importance of considering the enantioselectivity of chiral contaminants in the

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 21607128, 21320102007, 21277126) and the Zhejiang Provincial Natural Science Foundation of China (No. Y5090252). We thank Dr. Jay Gan for his suggestion.

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