A bioenergetics assay for studying the effects of environmental stressors on mitochondrial function in vivo in zebrafish larvae

Abstract

Mitochondria, an integral component of cellular energy metabolism and other key functions, are extremely vulnerable to damage by environmental stressors. Although methods to measure mitochondrial function in vitro exist, sensitive, medium- to high-throughput assays that assess respiration within physiologically-relevant whole organisms are needed to identify drugs and/or chemicals that disrupt mitochondrial function, particularly at sensitive early developmental stages. Consequently, we have developed and optimized an assay to measure mitochondrial bioenergetics in zebrafish larvae using the XF^e24 Extracellular Flux Analyzer. To prevent larval movement from confounding oxygen consumption measurements, we relied on MS-222-based anesthetization. We obtained stable measurement values in the absence of effects on average oxygen consumption rate and subsequently optimized the use of pharmacological agents for metabolic partitioning. To confirm assay reproducibility we demonstrated that triclosan, a positive control, significantly decreased spare respiratory capacity. We then exposed zebrafish from 5 hours post-fertilization (hpf) to 6 days post-fertilization (dpf) to three polycyclic aromatic hydrocarbons (PAHs) – benzo(a)pyrene (BaP), phenanthrene (Phe), and fluoranthene (FL) – and measured various fundamental parameters of mitochondrial respiratory chain function, including maximal respiration, spare respiratory capacity, mitochondrial and non-mitochondrial respiration. Exposure to all three PAHs decreased spare respiratory capacity and maximal respiration. Additionally, Phe exposure increased non-mitochondrial respiration and FL exposure decreased mitochondrial respiration and increased non-mitochondrial respiration. Overall, this whole organism-based assay provides a platform for examining mitochondrial dysfunction in vivo at critical developmental stages. It has important implications in biomedical sciences, toxicology and ecophysiology, particularly to examine the effects of environmental chemicals and/or drugs on mitochondrial bioenergetics.

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 XF^e24 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 XF^e24 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.