Abstract:

Ultraviolet (UV) radiation is a common ecological factor that can greatly enhance the toxicity of crude oil and polycyclic aromatic hydrocarbons (PAHs), compounding exposure in aquatic environments. The gills of adult fishes may be the first tissue impacted by PAH exposure which could compromise gill epithelial tissue function including gas diffusion and osmoregulation. To investigate the impacts of UV enhanced PAHs on fish gills, adult Sheepshead minnows were exposed to anthraquinone (a photo-modified PAH; 40 µg/L dissolved in Dimethyl sulfoxide (DMSO)) for 48-hours. Following exposure Mitochondrial respiration was measured in extracted gill filaments using an Oroboros Respirometer. We hypothesized mitochondrial function would be compromised and manifest as decreased respiration in conjunction with increased reactive oxygen species (ROS) production. Minnows were exposed for 48-hours to anthraquinone dissolved in Dimethyl sulfoxide.

Suggested Citation:

Reynolds, A., Crossley, D., Roberts, A.. 2019. Simultaneous oxidative phosphorylation (OXPHOS) and reactive oxygen species (ROS) production of Sheepshead minnows exposed to anthraquinone. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N71Z4304

Publications:

Kirby, A. R., Galli, G., Crossley, J., Sweet, L. E., Crossley, D. A., & Roberts, A. P. (2020). Gill filament permeabilization: A novel approach to assess mitochondrial function in sheepshead minnows (Cyprinodon variegatus) following anthraquinone exposure. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 230, 108699. doi:10.1016/j.cbpc.2019.108699

Purpose:

To assess mitochondrial function following a 48-hour exposure period to anthraquinone (a photo-modified PAH).

Data Parameters and Units:

Data files for this experiment are organized into two folders: 1) Raw Oroboros Files exported to .csv and 2) Simultaneous ROS and Respiration Data Imported from .csv files. Raw Oroboros Files exported to .csv: contains output data from the Oroboros respirometer (Oroboros Instruments; Innsbruck, Austria). The filenames contain the date of the experiment, “cont” means control, “aq” mean anthraquinone exposed minnows, “part 1” means the file contains data for Respiration (cytochrome c test and complex IV), “part 2” means the file contains data for simultaneous ROS (reactive oxygen species) measurements. The control and exposed are in the same file because the data were recorded simultaneously from two chambers (one being the control chamber and the other being the exposed chamber). On June 05 there was only 1 chamber working so the protocol was broken up. The filename contains also the exposure time which is 48h for all. Please note that there is a typo in some of the filenames which say 24h, this should be 48h. If a filename has the word “partial” it means the data file crashed halfway through data recording and so this file is partial/incomplete data file. Below a list of all the files and the corresponding experiment name or number used for the data analysis (files in folder “Simultaneous ROS and Respiration Data Imported from .csv”). We ran a total of 16 control and exposed animals but narrowed the results to my best 8 trials (list below with the experiment name). However, the dataset includes all 16 experiments. LaurenRep: 2016-10-26_OXPHOS_C8_AQ8_GILL PERM FIBERS.csv LaurenRep: 2016-10-26_OXPHOS_ROS_C8_AQ8_GILL PERM FIBERS.csv Rep1: 2017_2MAY_CONT_ AQ_24HR_EXPOSURE_PART1.csv Rep1: 2017_2MAY_CONT_ AQ_24HR_EXPOSURE_PART2.csv Rep2: 2017_3MAY_CONT_ AQ_24HR_EXPOSURE_PART1.csv Rep2: 2017_3MAY_CONT_ AQ_24HR_EXPOSURE_PART2.csv Rep6: 2017_11MAY_CONT_ AQ_48HR_EXPOSURE_PART1.csv Rep6: 2017_11MAY_CONT_ AQ_48HR_EXPOSURE_PART2.csv Rep7: 2017_23MAY_CONT_AQ_48HREXPOSURE_PART1.csv Rep7: 2017_23MAY_CONT_AQ_48HREXPOSURE_PART2.csv Rep9: 2017_25MAY_CONT_AQ_48HREXPOSURE_PART1.csv Rep9: 2017_25MAY_CONT_AQ_48HREXPOSURE_PART2.csv Rep10: 2017_05JUNE_CONT_AQ_48HREXPOSURE_PART1.csv Rep10: 2017_05JUNE_CONT_AQ_48HREXPOSURE_PART2.csv Rep11: 2017_06JUNE_CONT_AQ_48HREXPOSURE_PART1.csv Rep11: 2017_06JUNE_CONT_AQ_48HREXPOSURE_PART2.csv Simultaneous ROS and Respiration Data Imported from .csv files: contain all the data files that were used for the data analysis in this study. Part I and Part II are the same as in folder one (i.e. part I, respiration and part II, ROS measurement). The headings for all the files in this folder are used as an indication of where we averaged the measurement for respiration rate in mitochondria. So, in the case of substrates, those values are immediately following the addition of mitochondrial substrates (succinate, glutamate, and malate). The title for ADP is immediately following the addition of ADP to the mitochondria, and so forth. When measurements are made right after substrate (succinate, glutamate, and malate) addition then the heading is labeled “Substrate”. When the heading is labeled “leak respiration”, it means measurements were made after a substantial period of time has passed after the addition of substrate. Mitochondrial respiration was measured in pmol O2/second/mg wet weight fish. ROS production was measured in fmol O2 consumption/second/mg wet weight fish. The units for cytochrome c test are % increase in pmol O2/second/mg. "TMPD&Asc" = tetramethyl-p-phenylenediamine and ascorbate. RCR (respiratory coupling ratio) is unitless. ADP and Succinate units are pmol O2/second/mg wet weight of tissue.

Methods:

Adult Sheepshead minnows were exposed to anthraquinone (40 µg/L) for 48 hours. Following exposure Mitochondrial respiration was measured in extracted gill filaments.

Instruments:

Oroboros Respirometer (Oroboros Instruments, Austria).

Error Analysis:

Statistical test ANOVA

Individual Files: