Exposure to di-(2-ethylhexyl) phthalate reduces secretion of GDNF via interfering with estrogen pathway and downregulating ERK/c-fos signaling pathway in astrocytes

doi:10.1016/j.fct.2021.112592

Food and Chemical Toxicology

Volume 158, December 2021, 112592

https://doi.org/10.1016/j.fct.2021.112592 Get rights and content

Highlights

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Maternal DEHP exposure reduces the secretion of GDNF in male offspring.
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Maternal DEHP exposure inhibits the ERK/c-fos pathway in male offspring.
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DEHP decreases GDNF secretion by disturbing the estrogen binding to ERα.
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DEHP reduces GDNF levels by downregulating ERK/c-fos pathway in astrocytes.

Abstract

Di-(2-ethylhexyl) phthalate (DEHP) is a typical endocrine-disrupting chemical (EDC) that can increase the risk of central nervous system disease. This study aimed to investigate the in vitro and in vivo effects of DEHP exposure on GDNF secretion and the underlying mechanisms. Pregnant Wistar rats were randomly assigned into four groups and administered 0, 30, 300, or 750 mg/kg DEHP daily by oral gavage. In addition, primary astrocytes were exposed to mono-(2-ethylhexyl) phthalate (MEHP), the main metabolite of DEHP. Our results showed that DEHP exposure reduced GDNF levels and downregulated the ERK/c-fos signaling pathway in the cerebral cortex of male, but not female, offspring. Moreover, exogenous estrogen could overcome the decreased GDNF levels in astrocytes caused by MEHP exposure. MEHP also decreased p300 levels and downregulated the ERK/c-fos signaling pathway in primary astrocytes. Honokiol restored GDNF levels following MEHP exposure by activating the ERK/c-fos signaling pathway, while the inhibitor U0126 further reduced the GDNF levels. These results suggested that DEHP exposure could interfere with the normal effects of estrogen in the brain and downregulate the ERK/c-fos signaling pathway to decrease the GDNF secretion from astrocytes in the cerebral cortex.

Graphical abstract

Introduction

Di-(2-ethylhexyl) phthalate (DEHP) is an environmental endocrine-disrupting compound (Quintana-Belmares et al., 2018), which can increase the flexibility and strength of polyvinyl chloride used in food packages, medical devices, office supplies, and children's toys (Huff et al., 2018; Zhang et al., 2020). Because DEHP is not chemically bound to polyvinyl chloride (Zhang et al., 2018), its overuse causes extensive environmental exposure due to dust particles in the air and soil and its slow dissolution into groundwater (Rowdhwal and Chen, 2018). DEHP can also migrate directly into food from the packaging (Li et al., 2020). For the general population, DEHP exposure is mainly via food (Camacho et al., 2020; Hsu et al., 2020). Because DEHP poses a serious threat to food safety and human health, it has attracted the attention of the public and government management departments (Li et al., 2020).

DEHP exposure adversely affects reproductive health in mammals by acting as an estrogen-like compound (Sun et al., 2018). It also has potentially harmful effects (e.g., carcinogenicity and liver, kidney, and developmental toxicities) (Ban et al., 2014). In human epidemiological studies, DEHP showed an obesogenic effect (Schaedlich et al., 2018), and postnatal exposure to DEHP may dysregulate the immune response and induce childhood allergies (Yang et al., 2019). It is especially noteworthy that DEHP exposure can have an impact on the brain, including impaired neurodevelopment, behavioral problems, anxiety, depression, and altered stress responses (Seltenrich, 2018). Since DEHP can be detected in cord blood and placenta samples from pregnant women (Li et al., 2016), prenatal exposure to DEHP affects neurodevelopment and induces behavior syndromes in children (Chen et al., 2019).

Astrocytes are the most abundant glial cells in the central nervous system (Barakat et al., 2017). Astrocytes not only can support neurons but also regulate essential activities in the brain, such as the development of the central nervous system, modulation of the immune system, and maintenance of the blood-brain barrier (Ridet et al., 1997). Furthermore, astrocytes are generally believed to be targets for estrogen action (Hisaoka-Nakashima et al., 2015). As a typical environmental endocrine disruptors, DEHP can interfere with normal estrogen action by binding to estrogen receptors, adversely affecting neurodevelopment (Engel et al., 2017; Takeuchi et al., 2005; Zsarnovszky et al., 2005). Therefore, the effects of maternal DEHP exposure on astrocytes have raised concerns.

Astrocytes can produce various neurotrophic/growth factors (e.g., nerve growth factor [NGF], brain-derived neurotrophic factor [BDNF], and glial cell line-derived neurotrophic factor [GDNF]) (Ikeda et al., 1999; Kotliarova and Sidorova, 2021). These factors play a crucial role in neural plasticity (Allen and Barres, 2009) and support neurogenesis, gliogenesis, brain development, and survival (Hisaoka-Nakashima et al., 2015). GDNF is a neurotrophic factor involved in neuronal growth and survival (Ibanez and Andressoo, 2017; Ma et al., 2011; Mitroshina capital Ie et al., 2019). GDNF synthesis and secretion may be regulated by the estrogen signaling pathway (Zhu et al., 2013), contributing to the neuroprotective effects of estrogen (Morale et al., 2006). Estrogen can cross the blood-brain barrier (Rettberg et al., 2014) and activate the estrogen receptors (ERs) expressed on astrocytes to generate pleiotropic effects on brain function (Contreras-Zarate et al., 2019). ERs consist of two subtypes: ERα and ERβ. These receptors co-localize in various brain regions in rats and humans; however, ERα is the main ER in astrocytes (Dhandapani and Brann, 2002). The binding of estrogen to ERα can activate p300 (Liu et al., 2014), a histone acetyltransferases that catalyzes histone acetylation, a hallmark of gene activation (Li et al., 2017). P300 overexpression can upregulate extracellular regulated kinase 1/2 (ERK1/2) expression (Jin et al., 2019) and activate its downstream protein c-fos (Cui et al., 2011). The proto-oncogene c-fos is expressed in astrocytes (Rubio, 1997) and may regulate GDNF production (Koyama et al., 2004). Moreover, the mitogen-activated protein kinase (MAPK) signaling pathway plays a significant role in GDNF production (Gaesser and Fyffe-Maricich, 2016).

Although studies about the adverse effects of DEHP on the nervous system are increasing (Barakat et al., 2018; Du et al., 2017; Komada et al., 2016; Luu et al., 2017; Nuttall, 2017; Wu et al., 2014), there are few studies focused on the effects of DEHP on astrocytes. It has been reported that exposure to environmental chemicals can reduce the secretion of neurotrophic factors from astrocytes (Castro et al., 2015; Miyazaki et al., 2016). Therefore, the aim of our study was to explore the impacts of DEHP exposure on GDNF secretion in astrocytes and investigate the potential underlying mechanisms.

Section snippets

Animals

Female Wistar rats (230–250 g) were obtained from the Center for Experimental Animals at China Medical University (Shenyang, China) with National Animal Use License SCXK-LN2013-0007. All experiments and surgical procedures were approved by the Animal Use and Care Committee at China Medical University, which complies with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals used and their suffering. DEHP-free

Maternal DEHP exposure reduced GDNF levels and downregulated the ERK/c-fos signaling pathway in the cerebral cortex of male offspring

GDNF can be released by astrocytes and has been implicated in the development and maintenance of central and peripheral neurons (Fielder et al., 2018; Fujita et al., 2018). On PN7, GDNF levels in the cerebral cortex of male offspring were significantly decreased in the 300 and 750 mg/kg/day DEHP groups compared to the untreated controls (Fig. 1A and B, p < 0.05). Similarly, the GDNF levels in the male offspring of the 750 mg/kg/day DEHP group were less than in the 30 mg/kg/day DEHP group (Fig. 1

Discussion

There is a potential for extensive environmental exposure to DEHP due to its overuse; its metabolite MEHP can easily pass through the placental barrier and affect the development and function of the nervous system (Wojtowicz et al., 2019). Astrocytes play a vital role in the central nervous system (Kajitani et al., 2016) and are widely present in the cerebral cortex. Astrocytes can secrete many neurotrophic factors that are important for neuron repair, axon growth, and functional recovery (

Conclusions

In conclusion, maternal DEHP exposure decreased GDNF levels in the astrocytes of the cerebral cortex, which could be related to the downregulation of the ERK/c-fos signaling pathway. Our in vitro experiments further confirmed that MEHP exposure affected the estrogen pathway by competitively binding to ERα, resulting in reduced p300 activation and ERK phosphorylation and decreased GDNF secretion. Furthermore, honokiol restored the GDNF levels reduced by MEHP, whereas U0126 caused a further

CRediT authorship contribution statement

Jianan Wang: Investigation, Writing – original draft. Xudong Li: Methodology, Investigation, Formal analysis. Chaonan Wang: Investigation. Yan Li: Writing – original draft, All authors have read and agreed to the published version of the manuscript. Jinmiao Wang: Investigation. Rui Fang: Investigation. Jingsi Wang: Investigation. Jie Chen: Conceptualization. Jing Dong: Conceptualization, Methodology, Funding acquisition, Writing – review & 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.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (grant number 81472943) and the Natural Science Foundation of Liaoning Province, China (grant number 2019JH3/10300439).