Mitochondrial response and resilience to anthropogenic chemicals during embryonic development
Graphical abstract
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.
Section snippets
Exposure protocol
AB strain zebrafish embryos were collected and incubated at 28.5 °C in egg water (1 embryo/1 mL) until 1 hpf at which time embryos were screened for viability. They were then moved to treatment solutions (10 embryos per 10 mL) for 24 h at 28.5 °C. Embryos were treated with egg water supplemented with a given chemical as summarized in Table 1. These concentrations were determined via a literature search to determine environmentally relevant concentrations and low dose ranges that were previously
Chemical treatment specific mitochondrial toxicity profiles
Mitochondrial toxicity profiles for a given embryo were generated by injecting known mitochondrial inhibitors and an uncoupler during the eOCR measurements to the well containing an embryo that was previously treated with a chemical for 24 h. Data showed that every chemical that we tested, with the exception of naproxen sodium, had at least some statistically significant effect on embryonic mitochondria. TDCPP (0.1 μM) was found to have the greatest effect on mitochondrial eOCR by significantly
Discussion
The goal of our study was to develop a 96-well plate assay to elucidate vertebrate developmental mitochondrial effects of exposure to environmental contaminants, some of which are found in drinking water. We were able to deduce chemical impacts on early mitochondrial function via eOCR, given that cellular oxygen is primarily utilized by the mitochondrial electron transport chain during ATP synthesis. Further, by using Xfe96 Eflux technology we were able to subject embryos to specific ETC
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.
Acknowledgments
We thank Drs. Robert Wheeler, Julie Gosse, Rebecca Van Beneden and Sarah Conlin for their expertise and knowledge in this study. We thank Ms. Bradlyn McEttrick for her contribution to the above experiments. We also thank Mr. Mark Nilan for his care of the zebrafish and providing zebrafish embryos used in this study.
Funding
This work was supported by Agilent Technologies Inc. University Relations grant under the ACT-UR program project 4357; University of Maine Startup Funds to Dr. Nishad Jayasundara; University of Maine Interdisciplinary Undergraduate Research Grant through Research Reinvestment Funds.
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