Abstract:

This dataset contains raw oroboros data files, each showing the oxygen consumption traces and substrates added to the cardiac and red muscle mitochondria of red drum (Sciaenops ocellatus) after 24hrs OFS (oil from the surface) high energy water accommodated fraction (HEWAF) exposure.

Suggested Citation:

Johansen, J., Esbaugh, A.. 2019. Effects of oil exposure on red drum (Sciaenops ocellatus) cardiac and red muscle mitochondrial function. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/n7-y9mb-fn45

Publications:

Johansen, J. L., & Esbaugh, A. J. (2019). Oil-induced responses of cardiac and red muscle mitochondria in red drum (Sciaenops ocellatus). Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 219, 35–41. doi:10.1016/j.cbpc.2019.02.003

Purpose:

Examine the injury of oil exposure on cardiac and red skeletal muscle in marine fish.

Data Parameters and Units:

The folder "Raw oroboros data": Contains raw oroboros data files in DLD and CSV file formats, each showing the oxygen consumption traces and substrates added to the cardiac and red muscle mitochondria of red drum (Sciaenops ocellatus) after 24hrs OFS (oil from the surface) high energy water accommodated fraction (HEWAF) exposure. File naming convention for all the files is 'Redfish (oil concentration) (ID) (date)". The file “Mitochondria redfish –FINAL –Cont ALL DATA –Dec 2017.xlsx": Contains multiple worksheets; the worksheet "Mitochondria" includes- Sample ID, Exposure, Date, Time, pH, Temp, Sal, DO, Date, Time, pH, Temp, Sal, DO, Ammonia, Weight, TL, SL, Heart (mg), Muscle (mg), Cytochrome activity, Success?, Oil weight, Time start, Time in cone, Time out of cone, % HEWAF, Analyte sample (0 Hr, 24 Hr), 0Hr Oil name, and 24HR Oil name. The worksheets “Cardiac Control”, “Red Control”, “Cardiac 20%HEWAF”, ”RED 20%HEWAF”, “Cardiac 20%HEWAF” and “Red 40%HEWAF” includes - Complex I, Cyt C (Cytochrome C), OXPHOS (oxidative phosphorylation), OXPHOS with ADP (oxidative phosphorylation with adenosine diphosphate), LEAK, Max uncoupled ETS (Electron transport system), Inhibit Complex I, ROX (residual oxygen consumption), O2 (pmol/(s*mg)), mean, SD (standard deviation), n, and SE (Standard error), C1-C60 (Various tanks). The file TCT E039 -Mitochondria.xls: Contains multiple worksheets; the worksheet "Test condition table" includes - test condition parameters and values. The worksheet "Tank ID Dilution or Stock Code” includes - Tank ID Dilution or Stock Code, Start date, Start time, End date, End time, Nominal Treatment Concentration, Treatment Units, Replicate number, Notes, and Recorded by information. The worksheet "WAF Preparation” includes WAF Prep 1, WAF Prep 2, WAF Prep 3, …. WAF Prep 45. The worksheet "Water Quality Monitoring" includes - Tank ID Dilution or Stock Code, Date, Time, Period, Water temp. (C), "pH (S.U.)", D.O. (mg/L), Salinity (ppt), and Total ammonia (µM). The worksheet "Sample inventory" includes - Sampling date, Sampling time, Tank ID Dilution or Stock Code, Number organisms per sample, Unique ID Number, Sample ID, Description of Sample, Storage location, and Notes Recorded by information. Protocol = tissue examined (cardiac muscle or skeletal muscle); Sample type = Chamber used in the respirometer (chamber A or B); Cohort = tissue examined (cardiac muscle or skeletal muscle); Sample code = Fish ID; Sample concentration = amount of tissue per ml; Chamber volume = 2ml; Sample amount = amount of tissue in total; R1 = Voltage reading of oxygen saturated sample (control value); R0 = Voltage reading of oxygen-depleted sample (control value); Pb = partial pressure of oxygen in the atmosphere, measured in kPa; FM = calibration factor; O2 background a° = slope of oxygen depletion in the chamber at the beginning of the trial O2 background b° = slope of oxygen depletion in the chamber at the end of the trial; Start = time of start for measuring each parameter during the trial; Stop = time of end for measuring each parameter during the trial; N points = number of individual oxygen measurements completed during the trial period for each parameter. Parameters - Start = Start-Complex1 -leak state = background respiration of tissues within the chamber without addition of mitochondrial substrates; Complex 1 = ADP-Complex1-Phos state = tissue respiration after addition of catalase, hydrogen peroxide, malate, pyruvate, glutamate and ADP; Cyt c = Cyt C-Membrane integrity = tissue respiration after addition of cytochrome C; OXPHOS = Succ-Complex 1&2-Phos ste P = tissue respiration after addition of succinate; OXPOS with ADP = ADP-Complex1&2-Phos ste P = tissue respiration after addition of ADO; LEAK = Omy-LEAK L = tissue respiration after addition of oligomycin; Max uncoupled ETS = FCCP-Max uncoupled ETS E = tissue respiration after addition of FCCP; Inhibit Complex I = Rot -Complex1 Inhibit -Com2e = tissue respiration after addition of rotenone; ROX = AntiA-Complex2 inhibit ROX = tissue respiration after addition of antimycin A; Control ratios = subtraction or division between the parameters E (max uncoupled ETS), P (OXPHOS), L (Leak), ROX, cytochrome C change (Cyt c). Definition of all the abbreviation and other notes: HEWAF = high energy water accommodated fractions; PAH = Polycyclic Aromatic Hydrocarbons; Temperature = Centigrades = 22.5C; Time/recovery = 24hr; Exposure/Conc = 0% (0HEWAF), 20% (20HEWAF), 40% (40HEWAF) HEWAF = 0, 29.6ug/L or 64.5ug/L PAH; Species: Sciaenops ocellatus; Weight = mgram; Standard body length = Standard length in cm (SL); Total body length = total body length of fish in cm (TL); Tank and stock ID: C = control, T = oil exposure, (example T12); D.O.=Dissolved Oxygen; Sal = salinity of water in ppt; pH = pH of water; Tissue: Heart = cardiac mitochondria, Muscle = red skeletal muscle mitochondria;se = standard error; ADP = Adenosine diphosphate; ATP = Adenosine triphosphate ; Cyt c = cytochrome c; Succ=succinate; OMY = Oligomycin; FCCP = Carbonyl cyanide-4 (trifluoromethoxy)phenylhydrazone; Rot = Rotenone; Anti A = Antimycin A; Mal = Malate; Pyr = Pyruvate; Glu = Glutamate; h202 = Hydrogen peroxide.

Methods:

Mitochondrial function was assessed using an Oroboros Oxygraph 2k™ respirometer system (Oroboros Instruments, Innsbruck, Austria). Two Oroboros chambers were prepared with 2.0 ml MiR05 solution at 22.0 °C. The permeabilized cardiac and red skeletal tissues of a single individual were carefully blotted on an absorbent tissue and weighed on a 0.1 mg precision scale. A total of 5.4 ± 0.3 mg cardiac and 12.5 ± 0.5 mg red skeletal tissue (mean ± S.E.M.) were then placed into the respective test chambers. Respiration was measured as weight/specific O2 flux (pmol s/1 mg/1), calculated in real/time using the negative time derivative of O2 concentration and recorded in DatLab (Oroboros Instruments). Mitochondrial function and efficiency were assessed using the following substrate and solution injection sequences: a) 5uL catalase stock solution and 3.0 uL H2O2; b) 5.0 uL of 0.8 M malate; c) 5.0 uL of 2.0 M pyruvate; d) 10.0 uL of 2.0 M glutamate. These initial injections provided the fibers with complex I substrate in the absence of ADP, thereby allowing measurement of steady/state basal respiration in a non-phosphorylating leak state (CI-Leak). Complex I oxidative phosphorylation (CI-OXPHOS) was stimulated by adding 0.5 M ADP in 5uL steps until saturation. Cytochrome C (5 uL of 4 M stock) was then added to assess the integrity of the mitochondrial membrane. In cases where OXPHOS increased more than 10 %, the membrane integrity was deemed compromised and the protocol was terminated. Maximal respiration flux (OXPHOS) was assessed by adding Complex II substrate (20.0 uL of 1.0 M succinate) in addition to 5.0 uL of 0.5 M ADP to verify saturation. Respiration rate owing to the passive leak of protons across the mitochondrial membrane (LEAK) was assessed by injection of the ATP synthase inhibitor oligomycin (1.0 uL of 4.0 mg/ml stock). Uncoupled electron transport capacity (ETS) was assessed by adding the protonophore FCCP (1 M stock) in 0.5 uL steps until saturation. Contributions of complex II to ETS was assessed by inhibition of complex I via injection of 1.0 uL of 1.0 M rotenone. Finally, to control for other oxygen-consuming processes, remaining flux from the ETS (i.e. complex II and III) were inhibited via injection of 1.0 uL of 5.0 M Antimycin A (leaving only residual oxygen consumption, ROX).

Instruments:

Oroboros Oxygraph 2k™ respirometer system (Oroboros Instruments, Innsbruck, Austria).

Individual Files: