Gill filament permeabilization: A novel approach to assess mitochondrial function in sheepshead minnows (Cyprinodon variegatus) following anthraquinone exposure

https://doi.org/10.1016/j.cbpc.2019.108699Get rights and content

Highlights

  • Novel gill filament preparation to measure mitochondrial respiration and ros production.

  • Acute anthraquinone exposure increases respiration in oxphos and leak states.

  • Acute anthraquinone exposures decrease relative ros production in leak states.

  • Observed results may be evidence of mounting antioxidant defense response to xenobiotic molecules.

Abstract

Anthracene is a highly toxic polycyclic aromatic hydrocarbon (PAH), and its toxicity is increased 8-fold after compounding exposure to UV radiation. Exposure to either the parent or photo-modified compound has been shown to cause increases in reactive oxygen species (ROS) production and lipid peroxidation. Since the majority of ROS production occurs within mitochondria, we investigated simultaneous mitochondrial respiration and ROS production in the gills of sheepshead minnows (Cyprinodon variegatus) acutely (48 h) exposed to anthraquinone (40 μg l1). Anthraquinone exposure caused a 25% increase in oxidative phosphorylation with electrons donated to Complex I (OXPHOSCI) and a 33% increase in Leak respiration with oligomycin (Leak-OmyCI). ROS production was slightly increased (33.3%) in Leak state with oligomyocin respiring on Complex I substrates (Leak-OmyCI) after anthraquinone exposure, but this value remained unchanged in all other respiratory states. When ROS production was normalized to mitochondrial oxygen consumption, we found that ROS production was decreased in all respiratory states, but most noticeably in the Leak state. We speculate that differences in the antioxidant defense system may have played a role in decreased ROS production. Overall, in this paper we present a novel technique to measure mitochondrial function in the gill filaments of teleost fish exposed to xenobiotic molecules, and we show anthraquinone exposure alters aspects of oxidative phosphorylation and ROS production.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are a carcinogenic compound found in smoke, tar, and crude oil. PAHs are released into the atmosphere by the burning and distilling of coal ore (Dipple, 1985), and into aquatic environments through the harvesting and transportation of crude oil (Pasparakis et al., 2019). Some PAHs contain photodynamic properties that are responsive to wavelengths in the UVA spectrum (315–400 nm) and can magnify toxicity in aquatic systems, resulting in photo-enhanced toxicity (Roberts et al., 2017). Photodynamic PAHs are excited by UVA light (380 nm), resulting in the formation of excited singlet and triplet state molecules. These molecules are highly reactive with their surrounding media and generate photo-modified PAHs (byproducts), as well as reactive oxygen species (ROS), which enhances overall toxicity leading to oxidative stress (Arfsten et al., 1996; Mallakin et al., 1999). The photo-modified PAHs are typically oxidized, resulting in higher water solubility and bioavailability to aquatic organisms than the parent PAHs. These molecules are highly reactive in tissues and attach to membrane lipids, which compromise cellular and organelle membrane integrity (Sikkema et al., 1994).

Without the influence of UV, anthracene is a highly toxic PAH with an LC50 of approximately 16.6 μg l-1, which is enhanced eight-fold (LC50 of 1.94 μg l−1) after co-UV exposure (Weinstein and Polk, 2001). Bluegill sunfish (Lepomis macrochirus) exposed to 12 μg l−1 of anthracene resulted in 100% mortality within nine hours of direct UV exposure (Bowling et al., 1983). The photo modification of anthracene (as a result of co-UV exposure) forms over 20 toxic byproducts (including anthraquinone), and generally the photoproducts are more toxic than the parent compounds (Mallakin et al., 1999). In the case of anthracene, 50% growth inhibition in aquatic duckweed (Lemna gibba) was achieved at a concentration of 1.0 mg l−1, while the same growth inhibition was observed using photo-modified anthracene at lower concentrations (0.1 mg l-1) (Huang et al., 1995). Photodynamic PAHs (anthracene) absorb UV radiation resulting in an excited energy state and oxidized photoproducts (anthraquinone). The energy released as the excited compound returns to the ground state and can be transferred directly to cellular O2 or membrane lipids (Roberts et al., 2017). This process of photosensitization has been proposed as the mechanism of toxicity (for PAHs and their photoproducts) through the production of reactive oxygen species (ROS), resulting in oxidative damage to tissues and membranes (Mallakin et al., 1999; Roberts et al., 2017; Weinstein and Polk, 2001). In fact, markers of oxidative stress (increased lipid peroxidation and ROS production) were found in bluegill sunfish liver microsomes after 60 min of exposure to anthracene (3.015 μg ml−1) and UV light (Choi and Oris, 2000).

Considering that ROS production occurs in the mitochondrial complexes, we wanted to measure the effects of anthraquinone on mitochondrial function (oxygen consumption and coupling efficiency) while simultaneously measuring ROS production in sheepshead minnow (Cyprinodon variegatus) gill filaments. We chose the gill filament as a target tissue because PAHs easily flux across the gill epithelium and are rapidly dispersed into the bloodstream (Ramachandran et al., 2006). In the past, oxidative phosphorylation of the gills has been measured in purified isolated mitochondria. However, this preparation requires large tissue samples, which is not possible in small fish species, such as the sheepshead minnow. To this end, we have developed a novel, whole permeabilized gill filament preparation to assess mitochondrial function and simultaneous ROS production in sheepshead minnows following acute (48 h) anthraquinone exposure. Permeabilization of the tissue removes the influence of the antioxidant defense systems and cascade signaling and gives us access to the electron transport chain for pharmacological manipulations (Pesta and Gnaiger, 2012). Here we show that the permeabilized gill preparation is suitable for mitochondrial measurements and can be successfully utilized for toxicological investigations.

Section snippets

Animals

Adult, mix-sexed Sheepshead Minnows (Cyprinodon variegatus) were purchased from Aquatic Biosystems (Fort Collins, CO) and overnighted to the University of North Texas. Upon arrival, minnows were housed in 100 l tanks filled with 25 ppt saltwater maintained at 26 °C. Saltwater was mixed in the lab by combining deionized facility water with Instant Ocean® Sea Salt (United Pet Group, Blacksburg, VA). Minnows were kept on a 10:14 h light:dark photoperiod and fed pellet food (Skretting, Tooele, UT)

Results and discussion

To date, the effects of individual PAH exposure on intact mitochondrial function are relatively unknown. Few studies have highlighted the effects of crude oil exposure on mitochondrial function, which provided the framework for our investigation. We have developed a new preparation for measuring mitochondrial function in whole gill preparations from small fish. Furthermore, we have shown exposure to anthraquinone affects aspects of mitochondrial electron transport chain function and ROS

Declaration of competing interest

No conflicts of interest, financial or otherwise, are declared by the author(s).

Acknowledgments

This research was made possible by a grant from The Gulf of Mexico Research Initiative. Data are publicly available through the Gulf of Mexico Research Initiative Information & Data Cooperative (GRIIDC) at https://data.gulfresearchinitiative.org (doi: https://doi.org/10.7266/N71Z4304).

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