Mitochondrial response and resilience to anthropogenic chemicals during embryonic development

Highlights

• High throughput zebrafish assay to assess mitochondrial toxins during development.

• Contaminants induce non-monotonic and mitochondrial hormesis responses.

• After removal from a chemical treatment, some mitochondrial effects are reversible.

Abstract

Mitochondria are integral to maintaining cellular homeostasis. Optimum mitochondrial function is critical during embryonic development, as they play a key role in early signaling cascades and epigenetic programming, in addition to sustaining an adequate energy production. Mitochondria are sensitive targets of environmental toxins, potentially even at levels considered safe under current regulatory limits. Most mitochondrial analyses have focused only on chemical exposure effects in vitro or in isolated mitochondria. However, comparatively little is known about mitochondrial effects of chemical exposure during vertebrate embryogenesis, especially during the recovery phase following a chemical insult. Here, we used the zebrafish (Danio rerio), in a 96-well plate system, to examine mitochondrial effects of 24 chemicals including pharmaceuticals, industrial chemicals, and agrochemicals. We used oxygen consumption rate (OCR) during embryogenesis as a proxy for mitochondrial function. Embryonic OCR (eOCR) was measured in clean egg water immediately following 24 h of chemical exposure and subsequently for an additional 8 h. Each chemical, dependent upon the concentration, resulted in a unique eOCR response profile. While some eOCR effects were persistent or recoverable over time, some effects were only detected several hours after being removed from the exposure. Non-monotonic dose response effects as well as mitochondrial hormesis were also detected following exposure to some chemicals. Collectively, our study shows that mitochondrial response to chemicals are highly dynamic and warrant careful consideration when determining mitochondrial toxicity of a given chemical.

Introduction

Mitochondria are essential for eukaryotic cellular development and function. In addition to maintaining energy homeostasis, mitochondria are involved in a number of processes including oxidative stress response, reactive oxygen species (ROS) signaling, apoptotic signaling, Ca²⁺ signaling and regulation, and biosynthesis of macromolecules (Bhatti et al., 2017; Vakifahmetoglu-Norberg et al., 2017; Pathak and Trebak, 2018; Spinelli and Haigis, 2018; Pfanner et al., 2019). Importantly, mitochondria play a critical role in viability and survival of developing embryos (Mishra and Chan, 2014). Throughout embryogenesis, aside from ATP synthesis, mitochondria facilitate signaling cascades (Chandel 2014) and are involved in intracellular communication (Nagaraj et al., 2017). Mitochondrial synthesis of molecules such as acetyl CoA from glucose and NAD+ is important in acetylation and potential epigenetic modifications of the developing embryo (Moussaieff 2015). Consequently, developmental perturbations to mitochondria may have significant later-life health impacts (Xia et al., 2014., Lueng et al., 2013).

Mitochondria are highly sensitive to environmental toxin exposure. Mitochondrial function (e.g., oxidative phosphorylation) and mitochondrial structures (e.g., lipid membranes and mitochondrial DNA) are key targets of anthropogenic compounds such as agrochemicals, industrial chemicals, and pharmaceuticals (Meyer et al., 2018). Furthermore, xenobiotic chemicals may affect mitochondrial regulation of nuclear-cytoplasmic processes (e.g., hormonal activity) and signaling (e.g., ROS, apoptotic), as well as alter mitochondrial ATP synthesis to meet the increased energy demand.

Despite growing interest on mitochondrial toxins and their potential role in the etiology of chronic diseases, research is just beginning to emerge on mitochondrial effects of chemical exposure during embryonic development. Furthermore, current studies are typically conducted using cell culture assays and only primarily focus on acute mitochondrial effects (Nadanaciva et al., 2013). Significant limitations in these studies include lack of insight into putative effects of chemical metabolites derived in vivo from parent compounds, and persistent long-term effects.

Continuous discovery of persistent contaminants in the environment, such as PFOS (Perfluorooctanesulfonic acid; a perfluoroalkyl substance) in the drinking water (Hu et al., 2016), indicate the limitations in determining health effects of environmental exposures solely through chemical composition analyses. Therefore, effect-directed analyses of environmental samples can provide insights into long-term health consequences of environmental exposures. To this end, mitochondrial toxicity can be a useful end-point and provide broader insights into potential health outcomes of environmental exposures. For example, mitochondrial toxicity of drinking water samples may serve as an important indicator of overall water quality (Roubicek and de Souza-Pinto, 2017). Thus, higher throughput approaches assessing mitochondrial effects in a whole organismal exposure context, particularly during vulnerable life-history stages (e.g., embryogenesis) are highly valuable when screening for persistent cellular effects of an individual chemical and their mixtures.

Using zebrafish Danio rerio as a model, we optimized a 96-well plate mitochondrial function assay to determine embryonic mitochondrial response to contaminants. The zebrafish is a prominent developmental toxicology model (Nishimura et al., 2016) and has a rapid development time, in which major organ systems (e.g., the nervous system, cardiovascular system, kidneys, and liver) are beginning to form by 24 h post fertilization (Kimmel et al., 1995). This enables monitoring whole organismal level effects of exposure to chemicals and their metabolites. Additionally, zebrafish pharmacokinetic properties are highly conserved to that of humans and relevant in the context of monitoring human health (MacRae and Peterson, 2015; Garcia et al., 2016). Indeed, several previous studies have demonstrated the use of zebrafish embryos as a model for investigating chemical effects on whole embryo mitochondria (Stackley et al., 2011; Conlin et al., 2018; Sounders et al., 2018).

Expanding on these studies, the goals of the current study are three fold: (i) optimize a 96-well plate based assay to measure mitochondrial function during embryogenesis, (ii) elucidate mitochondrial effects of developmental exposure to chemical contaminants at environmentally relevant concentrations, and (iii) test the hypothesis that mitochondrial effects of chemical contaminant exposure, even at very low-levels, during early cellular development (cleavage, blastulation, gastrulation, and segmentation) will persist through maturation. Here, we measured embryonic oxygen consumption rate (eOCR) as a proxy for mitochondrial function following 24 h treatment with pharmaceuticals, agrochemicals and other industrially utilized chemicals, especially at concentrations found in aquatic systems. We focused on mitochondrial effects immediately following treatment with a chemical contaminant during the first 24 h of development. Subsequently, we also measured eOCR during an 8 h recovery period from the exposure to determine persistent mitochondrial effects of chemical contaminants. Overall, we present a 96-well plate assay to screen for mitochondrial toxins in an organismal developmental context.