ELSEVIER
Journal of Hazardous Materials
journal homepage: www.elsevier.com/locate/jhazmat
HAZARDOUS MATERIALS
*
Stereoisomeric selectivity in the endocrine-disrupting potential of cypermethrin using in vitro, in vivo, and in silico assays
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Quan Zhang ª,”, Shuqing Yuª, Xiaoyang Chenb, Lili Fuª, Wei Daiª, Sijia Guª
a Key Laboratory of Microbial Technology for Industrial Pollution Control of Zhejiang Province, College of Environment, Zhejiang University of Technology, Hangzhou, Zhejiang 310032, PR China
b Institute of Hydraulic and Environmental Engineering, Zhejiang University of Water Resources and Electric Power, Hangzhou, Zhejiang 310018, PR China
ARTICLE INFO
Editor: Dr. S. Nan
Keywords: Cypermethrin Endocrine-disrupting effects Stereoisomeric selectivity
ABSTRACT
Despite the ubiquity of cypermethrin (CYP) stereoisomers in environment biota, the stereoisomeric selectivity of endocrine-disrupting potency of a-CYP, ß-CYP, and 0-CYP has not been well studied. In this study, dual-luciferase reporter gene assays were adopted to analyze their potential endocrine-disrupting effects via four receptors (ERa, GRa, MR and RXR). The results showed that a-CYP was antagonistic to ERa, GRa, and MR with RIC20 of 9.1 x 10-7, 7.6 x 10-7, and 1.0 x 10-6 M, respectively. ß-CYP exhibited only ERa-mediated agonistic activity with a REC20 of 2.1 x 10-6 M. None of the CYP stereoisomers interacted with RXR. Molecular docking indicated that a-CYP had the strongest binding capacity to GR& among the compounds. The expression levels of steroid hormone-related genes in human adrenocortical carcinoma (H295R) cells displayed that all three compounds inhibited the transcription of 3-6HSD, indicating the block of turning cholesterol into different hormones. Both «-CYP and ß-CYP upregulated genes encoding estrogen- and aldosterone-forming enzymes including 17-ßHSD, CYP19, STAR, and CYP11B2. Mortality and malformation toxicity assays in zebrafish embryos revealed that the order of toxicity was a-CYP > ß-CYP > 0-CYP. Our results indicated that a-CYP may pose the strongest endocrine- disrupting effects. The data provided here will be helpful to systematically understand stereoisomeric selectivity in the endocrine-disrupting effects of cypermethrin.
1. Introduction
Cypermethrin (CYP) accounts for half of China’s total pyrethroid production (Zhuang et al., 2011) and is widely used in controlling pests in fields ranging from agriculture to public health (Ansari et al., 2011). After application, CYP can enter the aquatic ecosystem through agri- cultural penetration, runoff from the land, and enrichment in the food chain (Li et al., 2016). The concentrations of CYP range from 0.01 to 9.80 µg/L (~ 2.40 * 10-11 M ~ 2.35 * 10-8 M) in surface water, riparian drainage canals, and rainwater worldwide (Marino and Ronco, 2005; Guo et al., 2017). CYP can easily accumulate in sediment (Yuan et al., 2019) and is frequently detected in invertebrates and vertebrates
(Ansari et al., 2011). Notably, a high concentration of 4.24 µg/L (1.02
* 10-8 M) was reported in human breast milk from a malaria-control
area (Bouwman et al., 2006; Ana et al., 2017). The concentration
reached up to 31 ug/ml ( 7.45 * 10-5 M) in blood samples of
field-spraying workers, demonstrating a high risk to organisms,
including humans (Azmi and Naqvi, 2011). Due to its ubiquitous pres-
ence in the environment and organisms, it is urgent to pay attention to
the potential health risks of CYP.
There have been numerous studies on the toxicity and risks of CYP. Aquatic toxicity can induce oxidative stress, DNA damage, apoptosis, and developmental toxicity in zebrafish (Jin et al., 2011; Shi et al., 2011). CYP also has negative impacts on cerebral and hepatic tissue in
Abbreviation: CYP, cypermethrin; «-CYP, alpha-cypermethrin; ß-CYP, beta-cypermethrin; 0-CYP, theta-cypermethrin; ERa, estrogen receptor «; GRa, glucocor- ticoid receptor «; MR, mineralocorticoid receptor; AR, androgen receptor; TR, thyroid hormone receptor; HPG, hypothalamic-pituitary-gonadal; T, testicular testosterone; DMSO, dimethyl sulfoxide; MTS, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium); CD-FBS, Charcoal- dextran treated FBS; DLR, Dual-Luciferase Reporter; YSE, yolk sac edema; PE, pericardial edema; CB, crooked body; LOEL, lowest observed effect level; VTG, vitellogenin; RXR, retinoid X receptor; REC20, 20% relative effective concentration; RIC20, 20% relative inhibitory concentration; H295R, human adrenocortical carcinoma cells; CHO, Chinese hamster ovary cell; MCF-7, human breast cancer cell.
* Corresponding author.
E-mail address: quanzhang@zjut.edu.cn (Q. Zhang).
https://doi.org/10.1016/j.jhazmat.2021.125389
rats (Giray et al., 2001). And it is well known that, as an endocrine-disrupting chemical (EDC), CYP could interfere with hor- mone biosynthesis and impair normal homeostatic control or repro- duction (Diamanti-Kandarakis et al., 2009). For instance, luciferase reporter gene assays demonstrated that CYP exhibits ER agonistic ac- tivity and androgen receptor (AR), glucocorticoid receptor (GRa), and thyroid hormone receptor (TR) antagonistic activity (Kojima et al., 2004; Sun et al., 2007; Du et al., 2010; Zhang et al., 2016a, 2016b; J. Zhang et al., 2016). Transcription patterns of many key genes in the hypothalamic-pituitary-gonadal (HPG) axis are affected by CYP in zebrafish (Guo et al., 2017; Zhang et al., 2017) and rats (Ye et al., 2017). The levels of serum and testicular testosterone (T) are markedly reduced in CYP-treated male mice, which might be due to impaired spermato- genesis (Wang et al., 2010). In epidemiological studies, the case history of field workers clearly implies that the degree of CYP exposure is related to disease outcomes such as liver and kidney dysfunction (Azmi et al., 2006). Altogether, CYP can interact with a number of hormone receptors, alter hormone synthesis, and adversely affect sexual devel- opment as well as normal functioning of different organ systems.
Previous evidence has shown that isomers of the same pesticides possess similar physical and chemical properties, but their stereo- selective biological characteristics might be distinct (Lewis et al., 1999). Isomer-selective degradation of CYP has been reported in soil (Yang and Ji, 2015) and sediment (Liu et al., 2004). Although studies have reported enantioselectivity of CYP toxicity in vivo, C. dubia (Liu et al., 2004), zebrafish (Xu et al., 2010), and tadpoles (Xu and Huang, 2017), and in vitro (Zhang et al., 2016a, 2016b; J. Zhang et al., 2016; Ji et al. 2019), the application of pure enantiomer pesticide in the field is still a chal- lenge due to the cost of separation. In fact, «-CYP, ß-CYP, and 0-CYP, three stereoisomers, have been sold on the market and applied in the field in place of their enantiomers. However, little is known about the toxic effects of a-CYP, B-CYP, and 0-CYP, let alone for the differences in stereoisomeric selectivity between them. Only a few studies have shown that a-CYP, -CYP, and 0-CYP exert toxicity (Hartnik and Styrishave, 2008; Tian et al., 2009; Zhuang et al., 2011; Zhang et al., 2017; Lu et al., 2021) on oxidative metabolism, biotransformation, and reproduction in nontarget invertebrates and vertebrates.
In this study, we selected a-CYP, B-CYP, and 0-CYP to investigate the stereoisomeric selectivity in potential endocrine disruption by in vitro, in vivo, and in silico assays. Following the Environmental Protection Agency published guidelines for testing potential endocrine disrupting chemicals, a dual-luciferase reporter gene assay was adopted to examine their affinity for ERa, GRa, MR, and RXR in CHO cell lines. The results of the reporter gene assay were confirmed by molecular docking. The effect on steroid hormone synthesis was explored in H295R cells, which has proven its value as an effective screening tool (Zhang et al., 2005). Finally, the developmental toxicity of three CYP stereoisomers on zebrafish embryos was examined. Our results characterize the potential endocrine-disruption effect and present new information for risk assessment of «-CYP, ß-CYP, and 0-CYP.
2. Materials and methods
2.1. Chemicals and reagents
Alpha-cypermethrin («-CYP, CAS:67375-30-8), beta-cypermethrin (B-CYP, CAS:65731-84-2), and theta-cypermethrin (0-CYP, CAS:71697-59-1) were kindly provided by Zhejiang Academy of Agri- cultural Sciences. The cell culture reagents and detail chemical infor- mation are shown in Table S1.
2.2. MTS assay
The cytotoxicity test for a-CYP, ß-CYP, and 0-CYP in Chinese hamster ovary cell (CHO) was carried out with the CellTiter 96® AQueous Cell Proliferation Assay (MTS; Promega, Madison, WI). CHO cells were
cultured in 96-well plates at a density of 104 cells/well. The culture medium was replaced by the tested medium which contained 10-5~10-9 M of three CYP stereoisomers or 1 DMSO (negative control) and 176-estradiol (E2), hydrocortisone (HC), Aldosterone (AD), and 9-cis-retinoic acid (9-cis-RA, positive control) were added. Finally, following the instructions of the DLR assay (Promega, WI), both Firefly and Renilla luciferase activities were calcu- lated successively with a fluorescence spectrophotometer (Infinite M200, Tecan Trading AG, Switzerland). The activity was normalized to the internal control.
2.4. Quantitative real-time PCR (qRT-PCR)
H295R cells were seeded in 6-well plates at a density of approxi- mately 106 cells/well. After adhesion, cells were exposed to 10-5, 10-6 or 10-7 M of three CYP stereoisomers or 1 DMSO solution were set as the control and the solvent control group. Forty-eight zebrafish embryos were set for each group which were distributed randomly in 96-well plates placed at a temperature of 27 ± 1℃ with a 14:10 h day/night photoperiod. There were three repli- cates for each concentration. Morphological malformations include yolk sac edema (YSE) and Pericardial edema (PE). Arrows indicate morphological malformations observed in larvae.
Control
a.
B
0
12h
24h
48h
YŚE
96h
PE YSE
YSE
CYP19 is the only gene which encodes the aromatase required for E2 formation (Sanderson et al., 2002). Three CYP stereoisomers signifi- cantly induced 17ßHSD and CYP19, implying an elevated estrogen level in cells. Since CYP11B2 plays a key role in catalyzing the transformation of 11-deoxycorticosterone to aldosterone, the synthesis of aldosterone may be influenced by x-CYP and ß-CYP due to their ability to signifi- cantly upregulate CYP11B2. Data on CYP11B2 transcription suggested the weak potential for 0-CYP in cortisol synthesis disturbance.
Many studies have suggested that SPs are a class of EDCs which can interfere with development, exhibit developmental toxicity to aquatic organisms (Diamanti-Kandarakis et al., 2009). The developmental toxicity of zebrafish embryo may be very complex and partly due to the endocrine disruption effects of CYP. For example, researches have demonstrated that CYP could disrupt estrogen (E2) and T3 levels and expression of NRs genes that control feedback loops within the hypothalamic-pituitary-gonadal (HPG), hypothalamic-pituitary-thyroid (HPT), hypothalamic-pituitary-adrenocortical (HPA) axes, which caused the development toxicity in zebrafish embryos (Colborn et al., 1993; Guo et al., 2017; J. Zhang et al., 2018; Y. Zhang et al., 2018). Thus, we explored the three CYP stereoisomers’ potential endocrine disruption effecting to in vivo model, as the zebrafish embryo development may be highly related to variety of hormones and receptors (Ankley and John- son, 2004). The half-lives for elimination of several pyrethroids were greater than 48 h in aquatic vertebrates, and CYP may persist in the aquatic environment for 2-4 weeks (Ansari et al., 2011). ß-CYP was also reported to enhance VTG formation and E2 secretion, increase the numbers of spermatogonia and spermatocytes, and reduce the numbers of spermatids (J. Zhang et al., 2018; Y. Zhang et al., 2018). Aquatic vertebrates such as rainbow trout and common carp are therefore sus- ceptible to the presence of CYP (Das and Mukherjee, 2003; Shires, 2010; Yuan et al., 2019).
Endpoints of zebrafish embryo testing from this study showed that a-CYP, ß-CYP, and 0-CYP were extremely toxic to zebrafish. The LC50 values of a-CYP, B-CYP, and 0-CYP were between 10-9-10-8 M, 10-8-10-7 M, and greater than 10-7 M, respectively. Similar to previ- ous reports, our data revealed that the order of toxicity of these three
CYP stereoisomers to zebrafish embryos was «-CYP > ß-CYP > 0-CYP.
In conclusion, we evaluated the potential health risks of three CYP stereoisomers using in vitro, in vivo, and in silico assays. Our results showed that the potential toxicological effects were a-CYP > ß-CYP > 0-CYP. More investigations are required to clarify the potential health risks of commercially available agrochemical isomers.
CRediT authorship contribution statement
Quan Zhang: Conceptualization, Writing - original draft, Writing - review & editing, Funding acquisition, Supervision. Shuqing Yu: Methodology, Investigation, Writing - original draft, Sample Collection. Xiaoyang Chen: Writing - review & editing, Sample collection, Data analysis. Lili Fu: Writing - original draft, Data analysis. Wei Dai: Writing - review & editing, Data analysis. Sijia Gu: Writing - review & editing, Data analysis.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This study was supported by the Zhejiang Provincial Natural Science Foundation of China (LR21B070001) and National Natural Science Foundation of China (21777147).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jhazmat.2021.125389.
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