Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology
A bioenergetics assay for studying the effects of environmental stressors on mitochondrial function in vivo in zebrafish larvae
Introduction
Mitochondria are important organelles that regulate many critical biological processes, including ATP production, redox signaling, intracellular calcium signaling, and apoptosis (Dumollard et al., 2007, Meyer et al., 2013). Many human diseases, such as cancer, diabetes, and neurological, cardiovascular, and gastrointestinal disorders, have been associated with mitochondrial DNA (mtDNA) mutations or mitochondrial dysfunction (Ballinger, 2005, Chapman et al., 2014, Coskun et al., 2012, Dumollard et al., 2007, Rolo and Palmeira, 2006, Singh, 2006), and several drugs that cause numerous off-target mitochondrial effects have also been identified. Increasing evidence suggests that mitochondrial structure and function are highly vulnerable to damage by environmental contaminants. The high lipid content of mitochondria facilitates accumulation of lipophilic chemicals, and cytochrome P450 (CYP) enzymes found in mitochondria have the potential to activate previously nonreactive chemicals. Moreover, mitochondria lack some of the DNA repair mechanisms present for nuclear DNA (nDNA) damage repair and may accumulate mitochondrial DNA (mtDNA) mutations over time. Additionally, mitochondria constantly change their morphology, number, and composition depending on cell type, developmental stage, environmental cues, and metabolic demands, especially in response to environmental stressors (Meyer et al., 2013). Thus, there is a need to examine the mitochondrial effects of environmental chemicals, particularly for more sensitive early life stages, as many of these compounds may be undiscovered mitochondrial toxicants.
Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants formed from incomplete combustion of organic material. They originate from both natural and anthropogenic sources such as volcanoes, forest fires, cigarette smoke, and motorized vehicles (ATSDR, 1995). PAHs can enter aquatic ecosystems through soil erosion or runoff, atmospheric depositions, industrial effluent, or oil spills, and tend to accumulate in aquatic sediments over time (Cousin and Cachot, 2014). Due to urban expansion and increased use of automobiles, the concentration of these compounds in aquatic environments is steadily increasing (Lima et al., 2003). Potential adverse impacts of PAHs include carcinogenesis, effects on the reproductive, neurologic, and immune systems, and developmental abnormalities (Arkoosh and Kaattari, 1991, Brown et al., 2016, Hawkins et al., 1990, Incardona et al., 2011, Johnson et al., 1988, Van Tiem and Di Giulio, 2011, Vignet et al., 2014a, Vignet et al., 2014b). In fish, the most notable adverse developmental effects occur due to bioactivation of PAHs via the aryl hydrocarbon receptor (AHR) pathway, disrupting normal cardiovascular development and resulting in deformities such as elongated “stringy” hearts, impaired heart looping, decreased blood flow, and pericardial effusion (Billiard et al., 2006, Incardona et al., 2004, Van Tiem and Di Giulio, 2011, Wassenberg and Di Giulio, 2004).
Several studies indicate that mitochondria are an important target of PAH toxicity. This is not unexpected, as these very hydrophobic compounds tend to be attracted to lipid-rich mitochondrial membranes inside the cell (Eisler, 1987). PAHs and their metabolites have been shown to localize in mitochondria (Li et al., 2003), and a truncated form of CYP1A, the enzyme that predominantly metabolizes PAHs, is found in the inner mitochondrial membrane in mammals, suggesting that PAHs may be directly metabolized in the mitochondria (Addya et al., 1997). In addition, induction of mitochondrial CYP1A enzyme activity is observed in killifish following benzo(a)pyrene exposure (Jung and Di Giulio, 2010), and it has been demonstrated that bulky DNA adducts formed by PAH metabolites interact with mtDNA (Jung et al., 2009). Furthermore, exposure to PAHs in mammals is associated with effects on nDNA and mtDNA, changes in membrane potential, induction of apoptosis, decreases in ATP production, mitochondrial morphological changes and structural damage, and oxidative stress (Backer and Weinstein, 1980, Graziewicz et al., 2004, Li et al., 2003, Pavanello et al., 2013, Pieters et al., 2013, Xia et al., 2004, Zhu et al., 1995).
Traditionally, mitochondrial respiration has been assessed in vitro and in various animal models using the Clark-type oxygen electrode, which is limited to measuring one sample at a time and lacks sensitivity and throughput (Chance and Williams, 1955, Gruber et al., 2011, Stackley et al., 2011). However, recent technological advances, such as the development of the XFe24 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA), have allowed for a more streamlined method to analyze mitochondrial respiration using a 24- or 96-well microplate format. Despite significant advancements in development and application of assays that evaluate mitochondrial function in vitro and ex vivo (Jayasundara et al., 2015a, Tiernan et al., 2015, Wills et al., 2015), there is an ongoing need to develop medium- to high-throughput assays that rapidly assess mitochondrial function within whole organisms to ensure physiologically-relevant responses are measured. Therefore, the goals of this study were to 1) develop and optimize a reliable, robust assay using the XFe24 Extracellular Flux Analyzer and pharmacological agents to obtain in vivo measurements of mitochondrial respiratory chain parameters in zebrafish larvae, and 2) apply this assay to examine how developmental exposure to subteratogenic concentrations of three PAHs – benzo(a)pyrene, phenanthrene, and fluoranthene – affects normal mitochondrial function.
Section snippets
Animals
Laboratory-reared adult wildtype (5D) zebrafish (founder fish provided by Dr. David Volz, University of California, Riverside) were raised and maintained within a recirculating Aquatic Habitats® Z-Hab system (Pentair Aquatic Eco-systems, Inc., Apopka, FL, USA) containing conditioned reverse osmosis (RO) water (27–28 °C) on a 14 h:10 h light:dark cycle. Adult females and males were bred directly on-system using in-tank breeding traps suspended within 3-l tanks. For all experiments described below,
Optimization of MS-222-based anesthesia
To determine whether MS-222 affected basal OCR in 6 dpf larvae, we analyzed six independent plates containing 5–6 individual larvae per treatment per plate on the XFe24 Extracellular Flux Analyzer. Prior to addition of MS-222 at all concentrations, basal OCR measurements for an individual larva were extremely variable over time (Fig. 1A, 125 mg/l treatment shown). Average basal OCR before MS-222 injection was 257.4 pmol O2/min (75 mg/l), 250.3 pmol O2/min (125 mg/l), and 245.3 pmol O2/min (175 mg/l).
Discussion
The objectives of the current study were to 1) develop and optimize a bioenergetics assay to assess mitochondrial health in zebrafish larvae, and 2) apply this assay to assess mitochondrial toxicity of PAHs. Although a few assays have evaluated mitochondrial function in zebrafish embryos and larvae (Gibert et al., 2013, Grone et al., 2016, Kumar et al., 2016, Stackley et al., 2011), our assay adds significant value to these existing assays by using pharmacological agents to perturb metabolism
Conflict of interest statement
All authors declare no conflict of interest.
Acknowledgements
We gratefully thank Dr. David Volz for providing us with founder fish to establish our wildtype (5D) zebrafish colony. This research was supported by Duke University's Superfund Research Center (NIEHS P42-ES010356).
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