Triadimefon suppresses fetal adrenal gland development after in utero exposure
Graphical abstract
Illustration of the signaling pathway to regulate adrenal cortex development after triadimefon (TDM) exposure.
TDM increases the generation of ROS to cause the following actions: 1) down-regulating IGF1, which can activate AKT1 signaling for the growth of the adrenal gland; 2) down-regulating AT1R and MC2R, which can activate ERK1/2 for the growth and survival of the adrenal gland; 3) down-regulating NR5A1, which can activate steroidogenesis via up-regulating the expression of steroidogenesis-related genes; 4) activating the phosphorylation of AMPK, which is a negative signal to control adrenal steroidogenesis mainly via down-regulating Star, Cyp11b1, Cyp11b2 expression, eventually leading to reduced synthesis of aldosterone (ALDO) and corticosterone (CORT)
Introduction
Triadimefon is a broad-spectrum antifungal agent, which is widely used in agriculture to control mold and fungal infections. Due to its excellent antifungal activity, triadimefon has become the most important fungicide for many years. Triadimefon can cause multiple toxicities to the thyroid, nerve system, and reproductive system in mammalian models (Cao et al., 2017; Hester and Nesnow, 2008; Xi et al., 2012). It is considered an endocrine disruptor because it can disrupt steroid hormone metabolism in the gonads (testis) (Cao et al., 2017; Shen et al., 2017). Both gonads and adrenals can produce steroid hormones. In rats, adrenal glands mainly synthesize the glucocorticoid corticosterone (CORT) and mineralocorticoid aldosterone (ALDO). The inadequacy of the adrenal cortex to produce glucocorticoids and mineralocorticoids is called Addison’s disease (Soderbergh et al., 1996; Willenberg and Bornstein, 2000). Whether triadimefon exposure can cause adrenal steroid deficiency and its underlying mechanism remain unclear.
The adrenal gland contains a core area called the adrenal medulla, which secretes epinephrine and norepinephrine, and a cortical area called the adrenal cortex, which secretes steroids. In rat models, the adrenal cortex can be divided into several areas, each of which secretes different steroids. The outer zone is called the zona glomerulosa, which mainly produces ALDO (Willenberg and Bornstein, 2000). The inner area below zG is called the zona fasciculata (zF), which mainly synthesizes CORT (Willenberg and Bornstein, 2000). Both ALDO and CORT synthesis requires cholesterol as a substrate, which is taken up from circulation via scavenger receptor class B1 (SCARB1) that binds high-density lipoprotein (Zhou et al., 2018) or is formed via de novo synthesis. When cholesterol is in the cytoplasm, it is transported to the inner mitochondrial membrane after the regulation of steroidogenic acute regulatory protein (STAR) (Stocco, 2001) under the stimulation of the trophic hormone such as adrenocorticotropic hormone (ACTH) when it binds to ACTH receptor, MC2R (Lee et al., 2004). In the inner mitochondrial membrane, cytochrome P450 cholesterol side chain cleavage enzyme (CYP11A1) is located and catalyzes the production of pregnenolone from cholesterol (Chien et al., 2017). Mitochondria and microsomal 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) can catalyze pregnenolone to progesterone. In zG, cytochrome P450 steroid 21-hydroxylase (CYP21) and cytochrome P450 aldosterone synthase (CYP11B2) work together to synthesize ALDO (Vaidya and Dluhy, 2000). In the zF, CYP21 and cytochrome P450 11β-hydroxylase (CYP11B1) work together to synthesize CORT (Huang and Kang, 2019). CORT can be metabolized to 11-dehydrocorticosterone (11DHC) by 11β-hydroxysteroid dehydrogenase 2 (HSD11B2), which primarily exists in the zF and zG (Morita et al., 1997; Shimojo et al., 1996).
In a rat fetus, the adrenal glands begin to develop around gestational day (GD) 13 and have two tissues of different origins, namely the cortex and the medulla (Bibeau et al., 2010). They continue to grow after birth. The development of the adrenal cortex is regulated by a major transcription factor (NR5A1, also known as steroidogenic factor 1). When NR5A1 is mutated in mice, these mice show adrenal hypoplasia (Gut et al., 2005). The development of the adrenal gland is also regulated by growth factors and hormones. In zG, ALDO synthesis is mainly stimulated by angiotensin II receptor type 1 (AT1R, encoded by Atgr1), potassium, and ACTH via ACTH receptor MC2R (encoded by Mc2r) (Lee et al., 2004). In zF, CORT is mainly synthesized after the stimulation of ACTH through MC2R. Insulin growth factor I (IGF1) is also a key growth factor for the development of the adrenal cortex (He et al., 2017). In this study, we studied the effects of triadimefon exposure in utero on the development of the adrenal cortex and investigated the underlying mechanism.
Section snippets
Chemicals and animals
Triadimefon was purchased from Sigma (St Louis, MO). Trizol reagent kit was purchased from Invitrogen (Carlsbad, CA). Reverse transcription kit and SYBR green real-time quantitative polymerase chain reaction (qPCR) kit as well as BCA Protein Assay kit were obtained from Takara (Otsu, Japan). Male and female Sprague-Dawley rats (56 days of age) were purchased from Shanghai Laboratory Animal Center (Shanghai, China).
Animal treatment
Female Sprague-Dawley rats (age of 63 days) were mated with a known fertile male.
General parameters of toxicity
Female Sprague-Dawley dams were orally given 0, 25, 50, and 100 mg/kg/day triadimefon from GD 12 to GD 20. Before and after treatment, triadimefon did not change the weight and birth rate of the dams (Table S2). The number of fetuses per dam and the ratio of male/female fetuses did not change (Table S2). Triadimefon did not alter the body weight of the fetus (Table S2). No overt toxicity, including feed and water intake and behavior, was found. No dam or fetal death was found. This indicates
Discussion
Triadimefon is a widely used fungicide. Its potential risk to the human endocrine system has raised concerns. Here, we report that triadimefon significantly disrupted the development of fetal adrenal glands in male fetuses: 10-day in utero exposure to triadimefon markedly reduced serum ALDO, CORT, and ACTH levels, and down-regulated the expression of Atgr1, Mc2r, Cyp11b2, Igf1, Nr5a1 in the adrenal cortex of male fetuses and reduced the thickness of zF zone. Results herein showed that Atgr1 and
CRediT authorship contribution statement
Qiang Xu and Quanxu Chen: Conceptualization, Methodology, Animal experiment, Software, Data curation; Liben Lin: Investigation and analysis and validation, Animal experiment; Pu Zhang: Investigation; Zengqiang Li: Investigation; Yige Yu: Investigation; Feifei Ma: Investigation; Yingfen Ying: Investigation; Xiaoheng Li: Investigation; Ren-shan Ge: Conceptualization, writing and editing.
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.
Acknowledgment
This work was supported by NSFC (81730042 to R.G.), Department of Health of Zhejiang Province (11-CX29 to R.G.).
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