Real-time in vitro monitoring of the subcellular toxicity of inorganic Hg and methylmercury in zebrafish cells

Highlights

• AIE probes were used to monitor the impacts of Hg²⁺ and MeHg on zebrafish cells.

• Both Hg²⁺ and MeHg decreased the lysosome pH, but their mechanisms were different.

• Hg²⁺exposure increased the number of intracellular lysosomes and lipid droplets.

• MeHg exposure impaired the mitochondrial respiratory function.

• Hg²⁺ caused more lysosomal acidification than MeHg, but was less toxic on mitochondria.

Abstract

Mercury (Hg) is a prominent environmental contaminant and can cause various subcellular effects. Elucidating the different subcellular toxicities of inorganic Hg (Hg²⁺) and methylmercury (MeHg) is critical for understanding their overall cytotoxicity. In this study, we employed aggregation-induced emission (AIE) probes to investigate the toxicity of Hg at the subcellular level using an aquatic embryonic zebrafish fibroblast cell line ZF4 as a model. The dynamic monitoring of lysosomal pH and the mapping of pH distribution during Hg²⁺ or MeHg exposure were successfully realized for the first time. We found that both Hg²⁺ and MeHg decreased the mean lysosomal pH, but with contrasting effects and mechanisms. Hg²⁺ had a greater impact on lysosomal pH than MeHg at a similar intracellular concentration. In addition, Hg²⁺ in comparison to MeHg exposure led to an increased number of lysosomes, probably because of their different effects on autophagy. We further showed that MeHg (200 nM) exposure had an inverse effect on mitochondrial respiratory function. A high dose (1000 nM) of Hg²⁺ increased the amount of intracellular lipid droplets by 13%, indicating that lipid droplets may potentially play a role in Hg²⁺detoxification. Our study suggested that, compared with other parameters, lysosome pH was most sensitive to Hg²⁺ and MeHg. Therefore, lysosomal pH can be used as a potential biomarker to assess the cellular toxicity of Hg in vitro.

Introduction

Mercury (Hg) is one of the most toxic metals and there have been longstanding concerns over its severe risks. Methylmercury (MeHg) is a highly toxic Hg form and can be biomagnified along food chains, posing threats to the health of wildlife and human beings (Driscoll et al., 2013; Krabbenhoft and Sunderland 2013; Obrist et al., 2018). Hg has a high affinity for thiol groups (including those of several transcriptional factors), which are essential to almost all aspects of cellular function (Genchi et al., 2017; Usuki et al., 2017; Yin et al., 2008; Zalups 2000), thereby disturbing various organelles and biochemical processes. However, due to the complexity and diversity of forms of Hg in cells, the effects induced by Hg in organelles are still unclear. Earlier studies attempted to link the toxicity of metals with subcellular distributions in aquatic animals (Wallace et al., 2003; Wang and Rainbow 2006), and mitochondria and lysosomes have been shown to be the major targets of Hg in fishes (Barst et al., 2016; Barst et al., 2018; Khadra et al., 2019). Thus, the responses of these two organelles are of particular interest with regard to the toxicity of Hg.

Many studies have shown that lysosomes are the important organelle targets of Hg (Braeckman and Raes 1999; Lauwerys and Buchet 1972; Weiyue et al., 2011; Zhang et al., 2017b). Lysosomes are membrane-bound organelles, responsible for the degradation of intracellular substances and the recovery of metabolites and ions to maintain homeostasis (Luzio et al., 2007). They are vital for various important cellular processes such as endocytosis (Miksa et al., 2009) and autophagy (Tian et al., 2019). The acid environment (pH 4.5 – 5.5) of lysosomes is maintained by the proton pump V-ATPase (Kim et al., 2013; Lawrence and Zoncu 2019; Niu et al., 2017), and any small fluctuation of lysosomal pH can adversely affect the conditions of the cells. For example, Jiang et al. (2012) reported that a decrease of lysosomal pH by only 0.2 units could damage the proteolysis of phagocytes of macrophages. The acidic environment can activate the functions of acid hydrolases and other enzymes in lysosomes. On the contrary, abnormal lysosomal pH can cause dysfunction, which is closely related to many diseases such as cancer and neurodegenerative diseases (Davies et al., 1993; Izumi et al., 2003; Schindler et al., 1996). Hg has been found to accumulate in lysosomes, but there are few reports on the effects of Hg on lysosomal pH.Therefore, real-time monitoring of the pH changes in lysosomes during Hg exposure is of great significance for investigating the toxicity induced by Hg.

Mitochondria have also been proved to be important targets of Hg, which can disrupt mitochondrial membrane stability and affect ATP synthesis and reduce mitochondrial pH (Belyaeva et al., 2011a; Pal et al., 2012; Schumacher and Abbott 2017). Mitochondria are the power houses of cells and their main function is to generate energy in the form of ATP. In addition, mitochondria also undertake many other physiological functions, such as regulating the membrane potential, cell growth, and metabolism (Dias and Bailly 2005; Nie et al., 2005; Zorova et al., 2018). Hg can disturb the membrane potential and calcium homeostasis of mitochondria (Carratù and Signorile 2015). Besides, Hg may bind to glutathione (GSH) and deplete the storage of GSH, resulting in an increase of reactive oxygen species ROS (Farina et al., 2013; Kim and Sharma 2004a). Hg also disturbs the number and size of lipid droplets (LDs) (Shi et al., 2018; Ung et al., 2010), which are dynamic lipid-rich spherical organelles and play central roles in energy balance, lipid storage and metabolism (Thiam et al., 2013). Besides, lipid storage is essential to prevent the toxicity of ROS (Zhang et al., 2017a). Due to their role in lipid storage and metabolism, lipid droplets are mainly found in common diseases related to lipid accumulation, including obesity, diabetes and even cancer (Bozza and Viola 2010; Chowdhury et al., 2014). Hg exposure may also cause intracellular lipid accumulation and lipid metabolism disorder (Chauhan et al., 2019; Frontalini et al., 2016; Shi et al., 2018).Thus the location and concentration monitoring of LDs are also important in examinations of Hg subcellular toxicity.

Fluorescence techniques are now widely used in vivo detection and visualization because of their high selectivity and sensitivity, low cost, simplicity and non-invasive operation. Various fluorescent probes based on small molecules, polymer materials, nanoparticles and dosimeters have become powerful tools for biological research. Many fluorescent probes for lysosomes, mitochondria and LDs have been developed and commercialized for fluorescence imaging, such as LysoTracker, MitoTracker and Nile Red dyes. However, due to the aggregation induced quenching (ACQ) effect, these traditional probes can only be used in very dilute solution, which leads to poor photostability. This problem can be overcome by developing aggregation-induced emission (AIE) materials (Luo et al., 2001). AIE probes show no or very weak fluorescence in the dissolved state, but emit intensive fluorescence in the state of aggregation. They display superior merits of good biocompatibility, excellent photostability and specificity, and thus provide promising application as probes for cell imaging and real-time study of living cell dynamics. Herein, we have used three AIE probes to evaluate the toxicity of Hg²⁺ and MeHg for the first time. The novel AIE probes, CSMPP (Shi et al., 2020b), TPE-Ph-In (Zhao et al., 2015), TBP (Shi et al., 2020a), were used to monitor lysosomal pH, mitochondrial membrane potential, and lipid droplets in live zebrafish fibroblast cells during Hg²⁺and MeHg exposure. Furthermore, we quantified mitochondrial bioenergetics using Cell Mito Stress Test Kit. Our study has provided a new perspective for the assessment of cytotoxicity of Hg²⁺and MeHg by coupling various subcellular bioimaging and metabolic measurements.

In our study, we used the zebrafish fibroblast cell lines as the cell model. Cell lines are an important subject area in aquatic toxicology research, mainly because they do provide mechanistic understandings of cellular toxicity, which may not be able to be achieved by using whole animal systems. Our main objective was to develop a simple and non-invasive method for real-time monitoring of the toxicity of mercury in fish cells and identified the toxic mechanisms of mercury.