Methylmercury disrupts autophagic flux by inhibiting autophagosome-lysosome fusion in mouse germ cells

https://doi.org/10.1016/j.ecoenv.2020.110667Get rights and content

Highlights

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    Methylmercury exposure increased mouse germ cell apoptosis.

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    Methylmercury caused oxidative stress in germ cells.

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    Methylmercury induced mitophagy in germ cells.

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    Methylmercury inhibited the autophagic flux by impairing the lysosome.

Abstract

Methylmercury (MeHg) is an extremely toxic environmental pollutant that can cause serious male reproductive developmental dysplasia in humans and animals. However, the molecular mechanisms underlying MeHg-induced male reproductive injury are not fully clear. The purpose of this study was to explore whether mitophagy and lysosome dysfunction contribute to MeHg-induced apoptosis of germ cell and to determine the potential mechanism. First, we confirmed the exposure of GC2-spd cells to mercury. In GC2-spd cells (a mouse spermatocyte cell line), we found that MeHg treatment led to an obvious increase of cell apoptosis accompanied by a marked rise of LC3-II expression and an elevated number of autophagosomes. These results were associated with the induction of oxidative stress and mitophagy. Interestingly, we found that MeHg did not promote but prevented autophagosome-lysosome fusion by impairing the lysosome function. Furthermore, as a lysosome inhibitor, chloroquine pre-treatment obviously enhanced LC3-II expression and mitophagy formation in MeHg-treated cells. This further proved that the induction of mitophagy and the injury of the lysosome played an important role in the GC2-spd cell apoptosis induced by MeHg. Our findings indicate that MeHg caused apoptosis in the GC2-spd cells, which were dependent on oxidative stress-mediated mitophagy and the lysosome damaging-mediated inhibition of autophagic flux induced by MeHg.

Introduction

Methylmercury (MeHg) is a notorious poison that can cause persistent neurological dysfunction in animals and humans. Several studies have been performed to help reveal the pivotal mechanisms mediating MeHg-induced neurotoxicity for decades (Burbacher et al., 2015; Pierozan et al., 2017; Spulber et al., 2018). Previous studies showed that a low dose of MeHg exposure leads to neuritic degeneration and eventually causes abundant apoptosis of cerebrocortical neurons (Fujimura et al., 2009). Liu W et al. found that MeHg induced neurocyte injuries via disrupting the neuronal Ca2+ homeostasis and inhibiting the expression of N-methyl-D-aspartate receptors (NMDARs) in primary cultured cortical neurons (Liu et al., 2017). Worse still, more and more research has demonstrated that MeHg exposure had adverse effects on reproductive function and the reproductive outcome (Fuchsman et al., 2017; Fujimura and Usuki, 2020; Liu et al., 2016). ShinoHomma-Takeda et al. showed that methylmercury inhibited the expression of plasma testosterone and impaired germ cells through cell- and stage-specific apoptosis (Homma-Takeda et al., 2001). Our previous research demonstrated that oxidative stress and autophagy represented critical events related to the germ cell-toxic effects induced by MeHg (Chen et al., 2019). However, further studies are required to explore the molecular mechanisms underlying the MeHg-induced germ cell toxicity.

MeHg is known to evoke the production of reactive oxygen species (ROS), DNA damage, and cell death (de Oliveira Lima et al., 2018; dos Santos et al., 2018; Oliveira et al., 2020). The toxicity of MeHg depends largely on its electrophilic properties, which allow it to contact with the soft nucleophilic groups from low-molecular-weight biomolecules (Zhang et al., 2009). The interaction between MeHg and the soft nucleophilic groups from biomolecules is answer for the increased production of ROS and decreased antioxidant capacity (Aschner et al., 2010; Zhang et al., 2009). ROS are critical mediators of damage to the mitochondria, resulting in the injury of membranes and lipids, as well as nucleic acids and proteins. The production of ROS directly acting on the mitochondrial membrane proteins and lipids contribute to induction of apoptotic cascades by a dependent or independent mechanisms of mitochondrial permeability transition pore opening (Jordan et al., 2003; Polunas et al., 2011). In turn, mitochondrial dysfunction induced by MeHg may cause a surge ROS by mitochondria, which will further aggravate cell apoptosis (Roos et al., 2012). Apoptosis and autophagy are two inseparable events that induce cell death.

Autophagy is an evolutionarily conserved pathway which plays a critical role in the cellular components degradation, including peroxisomes, non-functional proteins, and mitochondria. Mitophagy refers to the process of clearing mitochondria by autophagy (Lemasters, 2005). Mitochondria were first detected in the autophagosome in 1957 (Clark, 1957). Thereafter, mitophagy was considered to play a critical role in mitochondrial removal (Kanki et al., 2009; Narendra et al., 2010). Previous research demonstrated that genome-wide visual screening revealed Atg32—a mitochondrial receptor essential for mitophagy that localises to the mitochondria and directly mediates the selective removal of mitochondria (Okamoto et al., 2009). The most recent research indicates that the abundance of ROS and DNA damage contribute to mitochondria degradation in retinal pigment epithelium, resulting in a visual loss in old people (Hyttinen et al., 2018). It has been shown that MeHg exposure can also evoke autophagy both in vivo and in vitro (Fang et al., 2016; Lin et al., 2019). The induction of autophagy plays a vital catalytic role in MeHg-reduced viability and induced apoptosis in astrocytic (Yuntao et al., 2016). Interestingly, inhibition of autophagy with chloroquine or 3-methyladenine obviously decreased MeHg-induced astrocytes apoptosis ratio (Yuntao et al., 2016).

Methylmercury has been shown to closely relate to poor male reproductive outcomes (Tan et al., 2009). More and more studies have shown that autophagy plays very important role in cell survival and death. However, the underlying mechanism of autophagy in MeHg-induced germ cell toxicity is still undetermined. Here, we examine the effect of MeHg on the activity of autophagy in germ cell-2 and decipher its underlying toxic mechanisms; therefore, identifying the potential targets for reproductive protection against MeHg.

Section snippets

Cell culture and treatment

The GC2-spd cells (a mouse spermatocyte cell line) were cultured in DMEM culture medium (HyClone, SH30022.01 B), 5% foetal bovine serum (Hangzhou Evergreen Institute Biological Engineering, China) and 1% (v/v) penicillin/streptomycin (Beyotime Biotechnology, China) at 37 °C a humidified atmosphere of 5% CO2. At 60% confluence, the GC2-spd cells were administrated Methylmercury (Sigma, USA) (0, 0.5, 1, 1.5 μM) for 24 h. Chloroquine (Sigma, USA) dissolved to 10 μM with cell culture medium 1 h

MeHg induces apoptosis in GC2-spd cells

We found that MeHg can induce a large number of GC2-spd cell deaths. As shown in Fig. 1A, treatment of cells with 0.5 μM, 1 μM, and 1.5 μM MeHg for 24 h caused about 6%, 24%, and 54% reductions in cell viability, respectively. The IC50 value of MeHg on the growth inhibition of GC2-spd cells was 1.5 μM. Additionally, we treated GC2-spd cells with MeHg and measured the cell apoptosis with the Tunel assay. Our data showed that MeHg drastically increases GC2-spd cell apoptosis (Fig. 1B). Mitophagy

Discussion

Mercury and mercury-containing compounds have been applied in many areas, as diverse as vaccine preservation, extraction of gold, haberdashery, dental amalgams, and skin creams for thousands of years (Clarkson and Magos, 2006). Microorganisms methylate inorganic mercury in water sediments converting it into methyl mercury (Roos et al., 2012). Methylmercury accumulates and persists in food chain, especially in fish and raptors. (Roos et al., 2012). The consumption of fish and aquatic products

Funding

This work was supported by the Natural Science Foundation of Hubei Province [2017CFB117].

CRediT authorship contribution statement

Na Chen: Methodology, Software, Writing - original draft. Xiaofeng Tang: Visualization, Investigation, Methodology, Data curation. Zhaoyang Ye: Resources, Software. Shanshan Wang: Validation, Supervision, Resources. Xianjin Xiao: Conceptualization, Funding acquisition.

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.

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